LC-MS/MS-based methods for characterizing proteins
12292447 ยท 2025-05-06
Assignee
- Nuseed Nutritional Australia Pty Ltd (Victoria, AU)
- Commonwealth Scientific And Industrial Research Organisation (ACT, AU)
Inventors
- Susan MacIntosh (Saint Paul, MN, US)
- Michelle Colgrave (Brisbane, AU)
- Keren Byrne (St. Lucia, AU)
- Jo Caine (Parkville, AU)
- Xue-Rong Zhou (Canberra, AU)
Cpc classification
H01J49/0036
ELECTRICITY
G01N30/7233
PHYSICS
International classification
Abstract
The embodiments herein provide methods to analyze the in vitro stability of recalcitrant or membrane-bound proteins in simulated gastric fluid (SGF) comprising the proteolytic enzyme, pepsin, and in combination with a novel pepsin-trypsin assay employing state-of-the-art mass spectrometric approaches, such as LC-MS/MS, to monitor the precise degradation products. The extent of protein digestion can be evaluated by the appearance of peptic products and the disappearance of tryptic peptide products (as a proxy for intact protein). The embodiments herein also provide methods for protein quantitation using high-sensitivity LC-MRM-MS quantification. The methods embodied herein are particularly useful in charactering proteins produced in transgenic plants, such as canola genetically engineered to produce long chain omega-3 polyunsaturated fatty acids.
Claims
1. A method for characterizing the stability of a target protein in a simulated digestion assay, comprising the steps of: (a) subjecting a target protein to pepsin digestion; (b) obtaining a plurality of samples of peptides resulting from pepsin digestion at a plurality of time points and collecting LC-MS/MS data directly for each of the plurality of pepsin digestion samples at each of the plurality of time points without first enriching for membrane or microsomal fractions or separating the target protein by electrophoresis; (c) subjecting the plurality of samples of pepsin digestion samples to complete trypsin digestion and collecting LC-MS/MS data directly for each of the plurality of pepsin-trypsin digestion samples after trypsin digestion is completed for each of the plurality of time points without first enriching for membrane or microsomal fractions or separating the target protein by electrophoresis; and (d) determining from the LC-MS/MS data the kinetics of target protein digestion and the susceptibility to proteolysis of specific regions of the target protein by comparing the data from each of the plurality of time points of the pepsin digestion with the data from the corresponding dual pepsin-trypsin digestion, wherein the decline of tryptic peptides obtained from the dual pepsin-trypsin digestion is used as a proxy for intact target protein, and the appearance and disappearance of peptic peptides is used to indicate the in vitro digestibility of the target protein.
2. The method of claim 1, wherein the target protein is a recombinant protein.
3. The method of claim 1, wherein the target protein is a recalcitrant or membrane-bound protein.
4. The method of claim 1, wherein the target protein is obtained from tissues or seeds of a transgenic plant.
5. The method of claim 1, wherein the target protein is obtained from transgenic Brassica.
6. The method of claim 1, wherein peptides liberated from said dual pepsin-trypsin digestion are assessed using protein extracts from a source selected from total protein extracts from canola or from recombinant proteins expressed in yeast, bacterial, or baculovirus expression systems.
7. The method of claim 1, wherein said dual pepsin-trypsin digestion is used to assess the allergenic potential of the target protein.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(48) All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
(49) As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, comprise, comprises and comprising are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term or is inclusive unless modified, for example, by either. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term about, which is generally 1% to +10%, depending on context as determined by one of skill in the art.
(50) Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
(51) The embodiments described herein provide novel techniques to quantify and characterize target membrane proteins using high sensitivity LC-MRM-MS. In particular, transgenic proteins of the 3LCPUFA synthesis pathway cloned into Brassica, and often associated with membranes in vivo, are currently not adequately characterized by antibodies as used, for example, in traditional western blot analysis. Regarding the characterization of protein content and function, solubilization (using detergents to replace the lipid of the membrane), and purification can increase levels of protein, but dissociation from membranes typically eliminates desaturase or elongase activity, likely due to a requirement for other proteins co-localized in the membrane, as well as cofactors, some as yet unknown. The present approach can be used to analyze recalcitrant proteins directly, without first enriching for membrane fractions, such as microsomes, or separating/enriching putative target proteins by electrophoresis, before employing the LC-MS/MS system. See Skinner et al., 64 J. Ag. Food Chem. 5251 (2016). Thus, in comparison with current LC-MS/MS detection, the present embodiments provide high throughput methods that enable analysis of many samples. Additionally, membrane or microsomal enrichment may exclude characterization of proteins that are not captured in a specific cell fraction. For example, debris discarded after initial centrifugation of cell lysates at 12 kg may contain target proteins: supposed ER protein, 4-desaturase, was detected in the 15 kg pellet and would have been excluded from the subsequent 100 kg microsomal pellet. Se Skinner et al., 2016.
(52) The method described herein was employed to characterize seven transgenic enzymesfatty acid desaturases and elongasesthat provided a biosynthetic pathway to convert oleic acid to DHA. More specifically, metabolic engineering of the 3LCPUFA, like eicosapentaenoic acid (EPA, 20:53) and docosahexaenoic acid (DHA, 22:63), in oil crops involved in the transgenic expression of several fatty acid desaturases and elongases in 3LCPUFA biosynthesis pathway. The transgenic enzymes (
(53) The functionalities and activities of these enzymes have been demonstrated in different heterologous expression systems including transgenic Arabidopsis, Camelina, and Brassica seeds. Petrie et al., PLoS One 7: e49165 (2012); Petrie et al., PLoS One 9: e85061 (2014). Based on the sequence similarity and functionality, these seven proteins can be classified into three groups, (1) yeast acyl-CoA type fatty acid desaturases including Lack1-12D and Picpa-3D that introduce a double bond at the 12 and 15 positions, respectively; (2) algae fatty acid elongases including Pyrco-6E and Pyrco-5E that add two carbons to the carboxyl end of fatty acids; and (3) algae front-end fatty acid desaturases that introduce a double bond between an existing double bond and the carboxyl end of fatty acids including Micpu-6D, Pavsa-5D and Pavsa-4D. Zhou et al., 2007. Although the engineered D-IA synthesis pathway genes were under the control of seed-specific promoters, other tissues in addition to seed were also assessed as described herein.
(54) The likelihood of allergic oral sensitization to a protein is first affected by the stability of the protein to gastrointestinal digestion. Astwood et al., 14 Nature Biotechnol. 1269 (1996). Full evaluation of each transgenic protein, including expression levels and protein stability, provides valuable information when assessing allergenic potential using a weight of evidence approach. These transgenic 3LCPUFA enzymes are tightly associated with membranes and expressed at low levels, however, hampering characterization by traditional means such as immunoassays. The present embodiments provide LC-MS/MS based methods to evaluate membrane proteins. An aspect of the present embodiments assesses the in vitro digestibility of the fatty acid biosynthesis enzymes introduced into 3LCPUFA-producing canola by digesting with pepsin.
(55) In vitro digestion models are used widely to assess the nutritional value of ingested proteins based on their amino acid bioavailability. The correlation between protein allergenicity and protein stability in an in vitro pepsin digestion assay has been reported previously. Astwood et al., 1996. When proteins are found to be highly digestible, the potential for systemic exposure is reduced. The current safety assessment strategy (Codex, 2003) is based on a weight-of-evidence approach recognizing that no single endpoint can predict human allergenicity potential. Based on this strategy, a number of factors are evaluated in the context of genetic plants: the gene source, determining the similarity of amino acid sequence of the newly expressed protein to known allergens, the abundance of the protein in the crop and the digestibility of the protein to in vitro digestion.
(56) Protein quantification by multiple reaction monitoring (MRM), using a triple quadrupole mass spectrometer, has been applied to clinical laboratory studies. Rauh, 883 J. Chromatog. B, 59 (2012); Gillette & Carr, 10 Nat. Meth. 28 (2013). Current analysis of proteins by MRM is based on detection of peptides derived from proteolytic digestion of the target protein, typically by trypsin. The measurement of tryptic peptides in a complex sample matrix may be achieved by adding a known concentration of an isotope-labelled peptide isomer as an internal standard (IS) to the sample before analysis. The labeled peptide isomer (referred to as heavy) contains an amino acid labelled with the stable isotopes, typically .sup.15N or .sup.13C, resulting in a mass increase compared to that of the native peptide isomer (referred to as light). When subjected to chromatographic separation, the heavy and light peptides show nearly identical elution profiles, allowing the detection of the light peptides (analytes) in the matrix background. When subjecting the peptides to MS/MS under conditions of collision-induced dissociation, the light and heavy peptides also undergo an identical fragmentation mechanism (transition) providing an additional level of quality control in confirming the peptide identity.
(57) Upon digestion with pepsin alone, there are a number of scenarios that may occur (
(58) By employing trypsin post-pepsin (see
(59) Thus, by examining the pepsin proteolytic fragments, the breakdown of a protein could be monitored, but it is noted that determining whether degradation had reached completion is a difficult task. To overcome this deficiency, the tryptic peptide products were used as a proxy for intact protein, wherein in the absence of pepsin, the amount of tryptic peptide was equivalent to 100% of protein being present. In the presence of pepsin (at varying time points during digestion), the level of tryptic peptides would be expected to decrease for peptides that contained a pepsin cleavage site. In this way the complete degradation of the protein can be monitored.
(60) In one specific embodiment, transgenic enzymes of the 3LCPUFA pathway were digested with pepsin for 0 minutes to 60 minutes, followed by complete trypsin digestion, while collecting samples at timed intervals. The decline of tryptic peptides was used as a proxy for intact protein, and the appearance and disappearance of peptic peptides was used to indicate the in vitro digestibility of transgenic proteins. Additionally, the level of tryptic peptide markers in a known quantity of total protein was quantified using a spiked internal standard (IS). By examining specific peptides (unique to the target transgenic proteins), this approach provides highly selective and sensitive measurement of the target membrane proteins.
(61) A similar principle employing LC-MS/MS was used to quantify each target protein in different plant tissues and seed. Applying the LC-MS/MS based method in transgenic plants demonstrated that seed-specific promoters correctly directed expression of transgenes only in developing and mature seed, in which seed the transgenic proteins were present at low levels (ng target/mg protein).
(62) More specifically, in some embodiments, calibration curves were generated in which the analyte concentration was varied, and a defined amount of IS was spiked into the standards. The response of the mass spectrometer was the integrated peak area for each MRM transition. The top three MRM transitions were summed (for both analyte and IS). The ratios of (summed analyte peak area)/(summed IS peak area) were plotted against the known analyte concentration. The endogenous peptide response was determined and the concentration interpolated from the calibration curve, thus allowing the quantification of the peptide as fmol target peptide per 100 g total protein. This value was converted to a ng equivalent per mg total protein based on the molecular mass of each target protein.
(63) Using protein extracts from a variety of sources including total protein extracts from canola and recombinant proteins expressed in either yeast, bacterial or baculovirus expression systems, the peptides liberated after tryptic digestion were assessed. The 3 LC-PUFA biosynthetic enzymes were thus characterized, allowing selection of peptides as biomarkers of each protein for quantification. The amino acid sequences of the enzymes are given in
(64) Protein extracts from a variety of sources including total protein extracts from canola, recombinant proteins expressed in either yeast, bacterial or baculovirus expression systems were used. The proteins were either provided in-solution or as excised gel slices. Gel bands were digested. See Byrne et al., 12 Proteomics 1 (2012). The solutions were subjected to filter-assisted sample preparation (FASP). Wisniewski et al., 6 Nature Meth. 359 (2009); Colgrave et al., 1370 J. Chromatog. A 105 (2014).
(65) Proteolytically digested proteins were analyzed with chromatographic separation using a nano HPLC system directly coupled to a mass spectrometer, and software used for protein identification. See, Shilov et al., 6 Mol. Cell Proteom. 1638 (2007). Tandem mass spectrometry data was searched against in silico tryptic digests of a database comprising the transgenic proteins, using parameters defined as iodoacetamide modified for cysteine alkylation and trypsin as the digestion enzyme.
(66) Total protein extracts from 3LCPUFA transgenic canola tissues or seed or from recombinant proteins expressed in yeast, bacterial, or baculovirus expression systems were first analyzed by non-targeted LC-MS for detection of the tryptic peptides generated for each target protein, e.g., desaturase or elongase, of the 3LCPUFA pathway. Total protein from Nicotiana benthamiana leaf with a transiently expressed marker protein was also used for detection of the tryptic peptides of the marker protein. After searching all generated data against the custom protein database, two peptides were selected from each target protein as proxies for use in quantification. The selection of peptides was based on the criteria: good MS response (high intensity); where possible, absence of amino acids within the peptide sequence that are likely to be modified (for example, oxidation of methionine) or miscleaved (presence of dibasic residues at either terminus); specific/unique to the target protein; and of a size amenable to LC-MS (6 to 20 amino acids in length). For each selected peptide, both the endogenous (light) peptides and .sup.15N and .sup.13C labelled (heavy) peptides were synthesized.
(67) The peptides liberated after tryptic digestion were assessed using protein extracts from a variety of sources including total protein extracts from canola or recombinant proteins expressed in either yeast, bacterial or baculovirus expression systems. The 3LCPUFA biosynthetic enzymes were thus characterized, allowing selection of peptides as biomarkers of each protein for quantification. See
EXAMPLES
Example 1. Peptide Selection
(68) Target ProteinsAll seven transgenic biosynthesis pathway enzymes expressed in 3LCPUFA canola were targeted for characterization, including quantification of protein content in various tissues of transgenic canola across the growing season.
(69) Using protein extracts from a variety of sources including total protein extracts from canola or recombinant proteins expressed in either yeast, bacterial or baculovirus expression systems, peptides liberated after tryptic digestion were assessed. The protein sequences are given in
(70) Selection of Peptides for QuantificationThe total protein extracts from DHA canola seed or from recombinant proteins expressed in either yeast, bacterial, or baculovirus expression systems were analyzed by non-targeted LC-MS for detection of the tryptic peptides generated for each target protein (i.e., desaturase or elongase of the 3LCPUFA biosynthesis pathway). Total protein from N. benthamiana leaf with transiently expressed R was also used for detection of the tryptic peptides of R protein. After searching all generated data against the custom protein database, two peptides were selected from each target protein as proxies to be used for quantification. The selection of peptides was based on several criteria: (a) good MS response (high intensity), (b) absence of amino acids within a peptide sequence likely to be modified (for example, oxidation of methionine) or miscleaved (presence of dibasic residues at either terminus), (c) amino acids specific/unique to the target protein, and (d) of a size amenable to LC-MS (6-20 amino acids in length). For each selected peptide, both the endogenous (light) peptides and .sup.15N and .sup.13C labelled (heavy) peptides were synthesized.
(71) Based on the preliminary results from both the quality control assessment and determination of linearity of response for the synthetic peptides, the peptide with optimal performance characteristics (e.g., high signal intensity, good chromatographic properties) for each protein was selected as the protein proxy for quantification. The final selected peptides for the DHA synthesis pathway enzymes (see
(72) TABLE-US-00001 TABLE 2 Peptides used for protein quantification of the DHA biosynthesis pathway enzymes Light Peptide MW Light MW Heavy Protein Sequence Peptide (Da) Heavy Peptide Sequence Peptide (Da) Lackl-12D GSSSNTEQEVPK.sup.1 1261.58 GSSSNTEQEV*PK.sup.1 1267.58 Picpa-3D IPFYHAR.sup.2 902.48 IPFYHA*R.sup.2 906.48 Micpu-6D DASTAPVDLK.sup.3 1015.52 DASTAPVDL*K.sup.3 1022.52 Pyrco-6E GQDPFLLK.sup.4 916.50 GQDPFLL*K.sup.4 923.50 Pavsa-5D AYDVTNFVK.sup.5 1055.53 AYDVTNFV*K.sup.5 1061.53 Pyrco-5E SQPFGLK.sup.6 775.42 SQPFGL*K.sup.6 782.42 Pavsa-4D LAPLVK.sup.7 639.43 LAPLV*K.sup.7 645.44 IS WEGEPI*SK.sup.8 951.46 Additional peptides for use in protein quantification: Lack1-12D GSSSNTEQEVPK.sup.1 1261.58 GSSSNTEQEVPK*.sup.1 1269.58 Lack1-12D NINNCGVGAAEK.sup.9 1189.3 NINNC.sup.[CAM]GVGAAEK.sup.9 1245.56 Lack1-12D NINNCGVGAAEK.sup.9 1189.3 NINNC.sup.[CAM]GVGAAEK*.sup.9 1253.56 Picpa-3D DILDAIPK.sup.10 883.50 DILDAIPK*.sup.10 891.50 Picpa-3D IPFYHAR.sup.2 902.48 IPFYHAR*.sup.2 912.48 Micpu-6D ALPSRPAEIK.sup.11 1081.29 ALPSRPAEIK*.sup.11 1088.63 Micpu-6D DASTAPVDLK.sup.3 1015.52 DASTAPVDLK*.sup.3 1023.52 Pyrco-6E GQDPFLLK.sup.4 916.50 GQDPFLLK*.sup.4 924.50 Pavsa-5D AYDVTNFVK.sup.5 1055.53 AYDVTNFVK*.sup.5 1063.53 Pyrco-5E SQPFGLK.sup.6 775.42 SQPFGLK*.sup.6 783.42 Pavsa-4D LAPLVK.sup.7 639.43 LAPLVK*.sup.7 647.43 Pavsa-4D WEGEPISK.sup.8 945.04 WEGEPISK*.sup.8 952.46 *Amino acid residues labeled with isotope .sup.15N or .sup.13C. Heavy peptides were used as reference standards for determining the correct retention time and fragmentation pattern. .sup.1GSSSNTEQEVPK: residues 16-26 of SEQ ID NO: 2; .sup.2IPFYHAR: aa 351-358 of SEQ ID NO: 1; .sup.3DASTAPVDLK: aa 30-39 of SEQ ID NO: 3; .sup.4GQDPFLLK: aa 83-90 of SEQ ID NO: 4; .sup.5AYDVTNFVK: aa 37-45 of SEQ ID NO: 5; .sup.6SQPFGLK: aa 66-72 of SEQ ID NO: 6; .sup.7LAPLVK: aa 403-408 of SEQ ID NO: 7; .sup.8WEGEPISK: aa 274-281 of SEQ ID NO: 7; .sup.9NINNCGVGAAEK: aa 405-416 of SEQ ID NO: 2, [CAM] is carbamidomethylated Cys; .sup.10DILDAIPK: (aa 51-58 of SEQ ID NO: 1); .sup.11ALPSRPAEIK: residues 06-115 of SEQ ID NO: 3.
(73) Synthesis of peptidesSelected peptides were synthesized at Creative Proteomics (Shirley, N.Y., US) at 90% c purity. The amount of each synthesized peptide was determined by high sensitivity amino acid analysis (AAA) at the Australian Proteomics Analysis Facility (Sydney, AU). All samples were analyzed in duplicate. The calculated amount of amino acid (g/ML) was based on the amino acid residue mass in the protein (molecular weight minus H.sub.2O). Using the determined concentrations, stock solutions were prepared at 100 pmol/L. The purity of synthesized peptides was analyzed by LC-MS. Dilutions equivalent to 5 pmol/L were prepared in aqueous solution (1% formic acid) and analyzed by LC-ESI-MS/MS. Any peptides showing significant contamination including the presence of the truncated, modified or synthesis by-products were excluded from further analysis.
(74) The purity of synthesized peptides was analyzed by LC-MS, and the results are shown in
(75) The concentrations of the synthetic peptides were determined by high sensitivity amino acid analysis and results were expressed as averages of duplicate measurements (Tables 3-11). The calculated amount of amino acid (g/mL) is based on the amino acid residue mass in the protein (molecular weight minus H.sub.2O). Using the determined concentrations, stock solutions were prepared at 100 pmol/L peptide.
(76) TABLE-US-00002 TABLE 3 Amino Acid Analysis: GSSSNTEQEVPK.sup.1 GSSSNTEQEVPK.sup.1 MW 1261.58 Da GSSSNTEQEV*PK.sup.1 MW 1267.58 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Ser 14.07 161.6 22.7 25.0 15.96 183.3 22.7 25.0 Gly 3.49 61.2 8.6 8.3 3.99 70.0 8.7 8.3 Asp 7.06 61.3 8.6 8.3 7.99 69.4 8.6 8.3 Glu 23.69 183.4 25.7 25.0 26.91 208.4 25.8 25.0 Thr 5.81 57.5 8.1 8.3 6.59 65.2 8.1 8.3 Pro 5.97 61.5 8.6 8.3 6.75 69.5 8.6 8.3 Lys 7.87 61.4 8.6 8.3 8.84 69.0 8.5 8.3 Val 6.44 65.0 9.1 8.3 7.30 73.7 9.1 8.3 Total 74.40 712.9 100.0 100.0 84.33 808.4 100.0 100.0 .sup.aSer, serine; Gly, glycine; Asp, aspartic acid; Glu, glutamic acid; Thr, threonine; Pro, proline; Lys, lysine; Val, valine; .sup.1GSSSNTEQEVPK: residues 16-26 of SEQ ID NO: 2.
(77) TABLE-US-00003 TABLE 4 Amino Acid Analysis: IPFYHAR.sup.1 IPFYHAR.sup.1 MW 902.48 Da IPFYHA*R.sup.1 MW 906.48 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % His 9.66 70.5 14.2 14.3 2.38 17.4 14.3 14.3 Arg 11.47 73.4 14.8 14.3 2.82 18.1 14.9 14.3 Ala 4.80 67.6 13.6 14.3 1.19 16.8 13.8 14.3 Pro 6.83 70.3 14.2 14.3 1.65 17.0 14.0 14.3 Tyr 11.58 71.0 14.3 14.3 2.83 17.3 14.3 14.3 Ile 8.00 70.7 14.3 14.3 1.93 17.1 14.1 14.3 Phe 10.60 72.0 14.5 14.3 2.58 17.5 14.5 14.3 Total 62.95 495.5 100.0 100.0 15.39 121.2 100.0 100.0 .sup.aHis, histidine; Arg, argininine; Ala, alanine; Pro, proline; Tyr, tyrosine; Ile, isoleucine; Phe, phenylalanine; .sup.1IPFYHAR: residues 351-358 of SEQ ID NO: 1.
(78) TABLE-US-00004 TABLE 5 Amino Acid Analysis: DASTAPVDLK.sup.1 DASTAPVDLK.sup.1 MW 1015.51 Da DASTAPVDL*K.sup.1 MW 1022.52 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Ser 5.47 62.9 8.9 10.0 3.44 39.5 9.0 10.0 Asp 16.50 143.4 20.4 20.0 10.47 91.0 20.6 20.0 Thr 6.86 67.9 9.7 10.0 4.30 42.6 9.7 10.0 Ala 9.73 136.9 19.5 20.0 6.08 85.6 19.4 20.0 Pro 6.92 71.3 10.1 10.0 4.34 44.7 10.2 10.0 Lys 9.22 72.0 10.2 10.0 5.66 44.2 10.0 10.0 Val 7.64 77.1 11.0 10.0 4.78 48.2 10.9 10.0 Leu 8.09 71.5 10.2 10.0 5.08 44.9 10.2 10.0 Total 70.45 702.9 100.0 100.0 44.16 440.6 100.0 100.0 .sup.aSer, serine; Asp, aspartic acid; Thr, threonine; Ala, alanine; Pro, proline; Lys, lysine; Val, valine; Leu, leucine; .sup.1DASTAPVDLK: residues 30-39 of SEQ ID NO: 3.
(79) TABLE-US-00005 TABLE 6 Amino Acid Analysis: GQDPFLLK.sup.1 GQDPFLLK.sup.1 MW 916.50 Da GQDPFLL*K.sup.1 MW 923.5 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Gly 4.34 76.2 12.6 12.5 2.70 47.3 12.4 12.5 Asp 8.75 76.0 12.6 12.5 5.53 48.0 12.6 12.5 Glu 9.84 76.2 12.6 12.5 6.11 47.3 12.4 12.5 Pro 7.33 75.4 12.5 12.5 4.62 47.6 12.5 12.5 Lys 9.58 74.7 12.3 12.5 6.15 48.0 12.6 12.5 Leu 17.01 150.3 24.8 25.0 10.79 95.3 25.0 25.0 Phe 11.22 76.2 12.6 12.5 7.04 47.8 12.5 12.5 Total 68.06 605.0 100.0 100.0 42.94 381.4 100.0 100.0 .sup.aGly, glycine; Asp, aspartic acid; Glu, glutamic acid; Pro, proline; Lys, lysine; Leu, leucine; Phe, phenylalanine; .sup.1GQDPFLLK: residues 83-90 of SEQ ID NO: 4.
(80) TABLE-US-00006 TABLE 7 Amino Acid Analysis: AYDVTNFVK.sup.1 AYDVTNFVK.sup.1 MW 1055.52 Da AYDVTNFV*K.sup.1 MW 1061.52 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Asp 15.38 133.6 22.3 22.2 15.02 130.5 22.3 22.2 Thr 6.32 62.6 10.5 11.1 6.18 61.2 10.4 11.1 Ala 4.48 63.0 10.5 11.1 4.36 61.4 10.5 11.1 Lys 8.41 65.6 11.0 11.1 8.24 64.3 11.0 11.1 Tyr 10.66 65.3 10.9 11.1 10.62 65.1 11.1 11.1 Val 13.92 140.4 23.5 22.2 13.59 137.1 23.4 22.2 Phe 9.97 67.7 11.3 11.1 9.77 66.4 11.3 11.1 Total 69.14 598.3 100.0 100 67.78 585.8 100.0 100 .sup.aAsp, aspartic acid; Thr, threonine; Ala, alanine; Lys, lysine; Tyr, tyrosine; Val, valine; Phe, phenylalanine; .sup.1AYDVTNFVK: residues 37-45 of SEQ ID NO: 5.
(81) TABLE-US-00007 TABLE 8 Amino Acid Analysis: SQPFGLK.sup.1 SQPFGLK.sup.1 MW 775.42 Da SQPFGL*K.sup.1 MW 782.42 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Ser 8.1 93 13.0 14.3 10.4 119 13.1 14.3 Gly 6.0 106 14.7 14.3 7.6 134 14.7 14.3 Glu 13.4 104 14.4 14.3 16.9 131 14.4 14.3 Pro 10.1 104 14.5 14.3 12.8 132 14.5 14.3 Lys 13.2 103 14.3 14.3 16.6 129 14.2 14.3 Leu 11.7 104 14.4 14.3 14.8 131 14.4 14.3 Phe 15.4 105 14.6 14.3 19.7 134 14.7 14.3 Total 78.0 718 100.0 100.0 98.8 909 100.0 100.0 .sup.aSer, serine; Glu, glutamic acid; Pro, proline; Lys, lysine; Leu, leucine; Phe, phenylalanine; .sup.1SQPFGLK: residues 66-72 of SEQ ID NO: 6
(82) TABLE-US-00008 TABLE 9 Amino Acid Analysis: LAPLVK.sup.1 LAPLVK.sup.1 MW 639.43 Da LAPLV*K.sup.1 MW 645.43 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Ala 8.13 114.3 16.0 16.7 4.69 66.0 15.8 16.7 Pro 11.63 119.7 16.8 16.7 6.71 69.1 16.6 16.7 Lys 15.19 118.5 16.6 16.7 8.78 68.5 16.5 16.7 Val 12.37 124.7 17.5 16.7 7.45 75.2 18.1 16.7 Leu 26.76 236.5 33.1 33.3 15.58 137.7 33.1 33.3 Total 74.07 713.8 100.0 100.0 43.21 416.4 100.0 100.0 .sup.aAla, alanine; Pro, proline; Lys, lysine; Val, valine; Leu, leucine; .sup.1LAPLVK; residues 403-408 of SEQ ID NO: 7.
(83) TABLE-US-00009 TABLE 10 Amino Acid Analysis of IS: WEGEPISK.sup.1 WEGEPISK.sup.1 MW 944.46 Da WEGEPI*SK.sup.1 MW 951.46 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Ser 6.6 76 12.8 14.3 6.8 78 13.0 14.3 Gly 4.9 86 14.4 14.3 5.0 88 14.8 14.3 Glu 22.1 171 28.8 28.6 22.1 171 28.7 28.6 Pro 8.4 87 14.6 14.3 8.4 87 14.5 14.3 Lys 11.3 88 14.8 14.3 10.9 85 14.2 14.3 Ile 9.8 87 14.6 14.3 9.9 88 14.7 14.3 Total 63.2 595 100.0 100.0 63.1 596 100.0 100.0 .sup.aSer, serine; Gly, glycine; Glu, glutamic acid; Pro, proline; Lys, lysine; Ile, isoleucine; .sup.1WEGEPISK: residues 274-281 of SEQ ID NO: 7.
(84) TABLE-US-00010 TABLE 11 Amino Acid Analysis of R Marker: TEPQTPQEWIDDLER.sup.1 TEPQTPQEWIDDLER.sup.1 TEPQTPQEWIDDL*ER.sup.1 MW 1855.86 Da MW 1862.86 Da Amino Amount Exp. Theor. Amount Exp. Theor. acid.sup.a (g/mL) nmol/mL mol % mol % (g/mL) nmol/mL mol % mol % Arg 6.12 39.2 7.3 7.1 4.90 31.4 7.5 7.1 Asp 9.43 82.0 15.3 14.3 6.98 60.6 14.5 14.3 Glu 25.1 194.4 36.4 35.7 19.35 149.9 35.8 35.7 Thr 7.12 70.4 13.2 14.3 5.75 56.9 13.6 14.3 Pro 7.15 73.7 13.8 14.3 5.74 59.1 14.1 14.3 Ile 4.16 36.8 6.9 7.1 3.32 29.4 7.0 7.1 Leu 4.25 37.6 7.0 7.1 3.50 31.0 7.4 7.1 Total 63.35 534.0 100.0 100.0 49.55 418.3 100.0 100.0 .sup.aArg, arginine; Asp, aspartic acid; Glu, glutamic acid; Thr, threonine; Pro, proline; Ile, isoleucine; Leu, leucine; .sup.1TEPQTPQEWIDDLER: SEQ ID NO: 8. An alternative peptide for use as a proxy of marker, R, expression in N. benthamiana is SVVAVIGLPNDPSVR (SEQ ID NO: 13), MW1521.85 (light) and SVVAVIGLPNDPSVR* (SEQ ID NO: 13), MW 1531.85 (heavy).
(85) As depicted in
(86) TABLE-US-00011 TABLE A Goodness of fit for quantitation of canola peptides (femtomoles) Protein Peptide Sequence LOD LLOQ ULOQ Sy.x SS R.sup.2 Lack1-12D NINNC[CAM]GVGAAEK 15.62 31.25 4,000 0.02143 0.01378 0.9996 Picpa-3D DILDAIPK 7.80 15.62 4,000 0.02249 0.17710 0.9997 Micpu-6D ALPSRPAEIK 7.80 15.62 4,000 0.08970 0.27360 0.9950 Pyrco-6E GQDPFLLK 7.80 15.62 4,000 0.02772 0.02767 0.9995 Pavsa-5D AYDVTNFVK 7.80 15.62 4,000 0.02695 0.02615 0.9988 Pyrco-5E SQPFGLK 7.80 15.62 4,000 0.02213 0.01764 0.9997 Pavsa-4D LAPLVK 7.80 7.80 4,000 0.03003 0.03608 0.9993 PAT SVVAVIGLPNDPSVR 7.80 31.25 4,000 0.00972 0.00302 0.9998 LOD, limit of detection; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; Sy.x, standard deviation of residuals, sum of the squares (SS); R.sup.2, non-linear regression, polynomial-cubic. Units in femtomoles loaded on-column.
Example 2. Sample Preparation
(87) All seven biosynthesis pathway enzymes expressed in the 3LCPUFA producing transgenic canola were targeted for quantification in various tissues and seed of transgenic canola throughout the growing season in separate planting locations.
(88) Collection of Canola SamplesWild-type (WT) and transgenic canola were planted at field trial sites. The tissues that were sampled for both WT and transgenic plants at each site are listed in Table B. The sampling times represent specific growth stages of canola allowing for various tissue types, including leaves, roots, pods, and reproductive tissues. See Lancashire et al., 119 Annals Appl. Biol. 561 (1991). The plant tissues harvested were maintained in dry ice to keep frozen, and transferred into 80 C. freezer until further processing.
(89) TABLE-US-00012 TABLE B Canola tissues sampled at different stages Growth stage Timing Tissue sampled Replicates TG15 5 True leaves 3 Whole plants 3 TG35 3 Visibly extended 1 Whole plant 3 internodes TG65 50% Full flowering All flowers from 1 plant 3 All roots from 1 plant 3 All leftover from 1 plant 3 TG79 Developing seed All pods form 1 plant 3 TG90 Senescence All grain from 1 plant 3
(90) Total Protein Extraction from CanolaThe collected samples (previously stored at 80 C.) were ground with mortar and pestle into fine powder with liquid nitrogen; all samples were maintained frozen on dry ice during the process. To avoid cross contamination, WT samples were processed first, then transgenic samples, in the order: TG15, TG35, TG65 root, TG other tissues, TG flower, TG79, and lastly TG90. Total protein was extracted from multiple aliquots of 100 mg in 2 mL plastic tubes in order to obtain more than 1 mg of total protein. Each tube was filled with 1 mL of 10% TCA in acetone and vortexed, then sonicated at frequency of 25% amplitude for 20 sec using a digital probe sonicator (Branson, St. Louis, Mo., US). Samples were centrifuged at 16,000g for 3 min at 4 C. The supernatant was removed by careful decanting. The pellet was resuspended in 1 mL of 0.1 M ammonium acetate (NH.sub.4CH.sub.3CO.sub.2) in 80% MeOH, mixed by vortexing, and centrifuged at 16,000g for 3 min at 4 C. The supernatant was discarded by careful decanting. The pellet was then resuspended in 1 mL 80% acetone, vortexed until the pellet was fully dispersed, and centrifuged at 16,000g for 3 min at 4 C. The supernatant was discarded, and the pellet air dried to remove the residual acetone.
(91) The air-dried pellet was re-suspended in 0.6 mL of UltraPure buffer-saturated phenol (Invitrogen, Carlsbad, Calif., US) and 0.6 mL freshly prepared SDS buffer (30% sucrose, 2% SDS, 0.1 M Tris-HC pH 8.8, 0.1 M DTT), mixed thoroughly, and incubated for 5 Min at room temp. The samples were then centrifuged at 16,000g for 5 min at room temp. The upper phenol phase was transferred to a new 2 mL tube, and 1 mL of 0.1 M NH.sub.4CH.sub.3CO.sub.2 in 80% MeOH was added. The proteins were precipitated at 20 C. overnight. Samples were centrifuged at 16,000g for 5 min at 4 C. The supernatant was carefully discarded, and the pellet was washed with 100% MeOH, then washed with 80% acetone. The proteins were pelleted by centrifuging at 16,000g for 5 min at 4 C. The final protein pellet was left to air dry.
(92) Canola Protein DigestionThe proteins extracted from different plant tissue or seed were dissolved in UA buffer (8 M urea, 0.1 M Tris-HCl, pH 8.5). Protein estimations were performed using a microtiter Bradford protein assay (Bio-Rad Labs., Hercules, Calif., US), following the reagent manufacturer instructions (version: Lit 33 Rev C). Samples were diluted in water over three dilutions, in duplicate, and measurements were made at 595 nm using a SpectraMax Plus. Bovine serum albumin (BSA) standard was used in the linear range 0.05 mg/mL to 0.5 mg/mL. The BSA standard concentration was determined by high sensitivity AAA at a commercial laboratory (Australian Proteomics Analysis Facility, Sydney, AU). Blank-corrected standard curves were run in duplicate. Linear regression was used to fit the standard curve.
(93) Protein SamplesProtein samples were stored at 80 C. prior to processing. Protein was subjected to FASP, wherein the protein extract (250 g) in UA buffer was applied to a 10 kDa molecular weight cut-off (MWCO) filter (Millipore, Sydney, AU) and diluted to 200 L with UA buffer before centrifugation (20,800g, 15 min). The protein on the filter was washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). To reduce the protein on the filter, DTT (100 mM, 100 L) was added and the solution incubated at room temp for 50 min with shaking. The filter was washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min).
(94) To alkylate the cysteine residues, iodoacetamide (IAM) (50 mM, 100 L) was applied to the protein on the filter with incubation for 20 min at room temp in the dark. The filter was washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was exchanged using 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) by two consecutive wash/centrifugation cycles. Then, 25 g sequencing grade porcine trypsin (Promega Corp., Alexandria, AU) (0.125 g trypsin/L in 200 L of 50 mM (NH.sub.4)HCO.sub.3, 1 mM CaCl.sub.2)) was added to the protein on the 10 kDa MWCO filters and incubated for 16 hr at 37 C. in a wet chamber. The filters were then transferred to fresh centrifuge tubes and the filtrate (digested peptides) collected following centrifugation (20,800g, 10 min). The filters were washed with 200 L of 100 mM (NH.sub.4)HCO.sub.3 and the filtrates combined and lyophilized. The resultant peptides were resuspended in 62.5 L of 1% formic acid containing 0.04 pmol/L of the IS peptide WEGEPI*SK (aa 274-281 of SEQ ID NO: 7) and 25 L (equivalent to 100 g of total protein and 1 pmol of IS) was analyzed by LC-MS/MS.
(95) Sample Preparation for LC-MS Method DevelopmentProtein extracts from a variety of sources, including total protein extracts from canola, recombinant proteins expressed in either yeast, bacterial or baculovirus expression systems, were tested. Proteins were either provided in solution or as excised gel slices. The solutions were subjected to FA SP as described previously. See Colgrave et al., 2014; Colgrave et al., 147 J. Proteom. 169 (2016). Gel bands were digested as described previously. Byrne et al., 2012.
Example 3. LC-MS Analysis
(96) Preliminary LC-MS AnalysisProteolytically digested proteins were analyzed with chromatographic separation (2%/min linear gradient of 2%-40% acetonitrile) using a nanoflow HPLC system (Prominence Nano, Shimadzu Corp., Rydalmere, Australia) directly coupled to a TripleTOF 5600+MS/MS system (AB S
(97) LC-MRM-MS QuantificationA series of standards (n=4 replicates) comprising a double blank (no analyte, no IS), a blank (IS only) and seventeen standards containing a known, but varied amount (0.08 to 5,000 fmol) of each peptide and 1 pmol of the IS peptide (WEGEPI*SK, aa 274-281 of SEQ ID NO: 7) were analyzed by LC-MRM-MS. The data were acquired using the Analyst 1.6.3 software on a QTRAP 6500+ LC-MS/MS system (AB S
(98) LC-MRM-MS Quantification of Canola ProteinsThe extracted and digested protein samples representing five growth stages (seven samples) from two growing sites, comprising both WT and transgenic canola (n=3 replicates, total 84 samples) containing the spiked IS were analyzed by LC-MRM-MS alongside aqueous peptide standards. An aliquot (25 L) of aqueous standard or canola peptide extract were chromatographically separated on a Nexera UHPLC (Shimadzu) and analyzed on a QTRAP 6500+ mass spectrometer. See Colgrave et al., 2014. Quantification was achieved using scheduled MRM scanning experiments using a 120 sec-detection window for each MRM transition and a 0.3 sec cycle time. Peaks were integrated using MultiQuant v3.0 (AB S
(99) Development of Quantitative LC-MRM-MS MethodUsing the data collected from the tryptic digests of the enzymes in the transgenic 3LCPUFA biosynthetic pathway, the peptide mass, and hence precursor mass-to-charge (m/z) ratio, was determined. Subsequently, five fragment ions were selected that were representative of the target peptide. Together, the Q1 m/z and Q3 m/z are termed the MRM transition, and the MRM transitions are presented in Table 12A for the light peptides, and Table 12B for the heavy peptides, heavy peptides were used as reference standards for qualitative assessment. Additional transitions information is in Table 13.
(100) TABLE-US-00013 TABLE 12A MRM transitions of light peptides (analytes) Protein Peptide RT Q1 m/z Z Q3 m/z Fragment CE 12D GSSSNTEQEVPK 1.62 631.797 2+ 729.380 y6+ 32.0 (aa 16-26 of SEQ 944.468 y8+ 28.0 ID NO: 2) 1019.428 b10+ 28.0 3D IPFYHAR 3.56 452.245 2+ 546.278 y4+ 27.2 (aa 351 358 of SEQ 693.347 y5+ 27.2 ID NO: 1) 395.703 y6++ 23.2 6D DASTAPVDLK 3.63 508.767 2+ 571.345 y5+ 23.9 (aa 30-39 of SEQ 642.382 y6+ 23.9 ID NO: 3) 743.430 y7+ 21.9 6 GQDPFLLK 5.07 459.258 2+ 260.197 v2+ 28.5 (aa 83-90 of SEQ 617.402 y5+ 21.5 ID NO: 4) 732.429 y6+ 19.5 5D AYDVTNFVK 4.85 528.772 2+ 608.34 y5+ 22.9 (aa 37-45 of SEQ 707.409 y6+ 22.9 ID NO: 5) 822.436 y7+ 22.9 5E SQPFGLK 3.84 388.719 2+ 216.098 b2+ 18.9 (aa 66-72 of SEQ 317.218 y3+ 26.9 ID NO: 6) 561.34 y5+ 18.9 4D LAPLVK 3.72 320.723 2+ 185.128 b2+ 14.0 (aa 403-408 of SEQ 246.181 y2+ 22.0 ID NO: 7) 456.318 y4+ 14.0 RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio; z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. Collision energy settings were also optimized for all targeted transitions by analyzing 2 L of each peptide chromatographically separated on a Nexera UHPLC and analyzed on a QTRAP 6500+ mass spectrometer. See Colgrave et al., 2014.
(101) TABLE-US-00014 TABLE 12B MRM transitions of heavy peptides (reference standards) including IS peptide Protein Peptide RT Q1 m/z Z Q3 m/z Fragment CE 12D GSSSNTEQEV*PK 1.62 634.79 2+ 735.380 y6+ 32.0 (aa 16-26 of SEQ 950.470 y8+ 28.0 ID NO: 2) 1025.420 b10+ 28.0 3D IPFYHA*R 3.56 454.245 2+ 550.278 y4+ 27.3 (aa 351 358 of SEQ 697.346 y5+ 27.3 ID NO: 1) 397.703 y6++ 23.3 6D DASTAPVDL*K 3.63 512.266 2+ 578.345 y5+ 23.9 (aa 30-39 of SEQ 649.382 y6+ 23.9 ID NO: 3) 750.429 y7+ 21.9 6 GQDPFLL*K 5.07 462.758 2+ 267.196 y2+ 27.7 (aa 83-90 of SEQ 624.402 y5+ 21.7 ID NO: 4) 739.429 y6+ 19.7 5D AYDVTNFV*K 4.85 531.771 2+ 614.340 y5+ 22.9 (aa 37-45 of SEQ 713.408 y6+ 22.9 ID NO: 5) 828.435 y7+ 22.9 SE SQPFGL*K 3.84 392.218 2+ 216.098 b2+ 24.9 (aa 66-72 of SEQ 324.218 y3+ 26.9 ID NO: 6) 568.339 y5+ 18.9 4D LAPLV*K 3.72 323.723 2+ 185.128 b2+ 14.0 (aa 403-408 of SEQ 252.181 y2+ 22.0 ID NO: 7) 462.318 y4+ 14.0 IS WEGEPI*SK 3.26 476.737 2+ 451.281 y4+ 28.0 (aa 274-281 of SEQ 637.346 y6+ 24.0 ID NO: 7) 766.388 y7+ 26.0 RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. Collision energy settings were also optimized for all targeted transitions by analyzing 2 L of each peptide were chromatographically separated by UHPLC and analyzed on a 6500 QTRAP mass spectrometer.
(102) TABLE-US-00015 TABLE 13 Additional Transitions Q1 m/z Q3 m/z Peptide Name CE 476.737 451.281 IS.XR2-d4D.WEGEPI*SK.+ 2y4 28 476.737 580.324 IS.XR2-d4D.WEGEPI*SK.+ 2y5 26 476.737 637.346 IS.XR2-d4D.WEGEPI*SK.+ 2y6 24 476.737 766.388 IS.XR2-d4D.WEGEPI*SK.+ 2y7 26 476.737 502.193 IS.XR2-d4D.WEGEPI*SK.+ 2b4 20 452.245 383.215 XR1-w3D.IPFYHAR. + 2y3 27.2 452.245 546.278 XR1-w3D.IPFYHAR. + 2y4 27.2 452.245 693.347 XR1-w3D.IPFYHAR. + 2y5 27.2 452.245 790.399 XR1-w3D.IPFYHAR. +2y6 23.2 452.245 395.703 XR1-w3D.IPFYHAR. + 2y6++ 23.2 442.76 244.17 XR1-w3D.DILDAIPK. + 2y2 20.7 442.76 428.29 XR1-w3D.DILDAIPK. + 2y4 20.7 442.76 528.27 XR1-w3D.DILDAIPK. + 2b5 20.7 442.76 543.31 XR1-w3D.DILDAIPK. + 2y5 20.7 442.76 656.4 XR1-w3D.DILDAIPK . + 2y6 20.7 320.723 185.128 XR2-d4D.LAPLVK. + 2b2 14 320.723 246.181 XR2-d4D.LAPLVK. + 2y2 22 320.723 359.265 XR2-d4D.LAPLVK. + 2y3 22 320.723 456.318 XR2-d4D.LAPLVK. + 2y4 14 320.723 527.355 XR2-d4D.LAPLVK. + 2y5 14 338.175 272.172 XR2-d4D.VHHGFYPR. + 3y2 18.2 338.175 582.304 XR2-d4D.VHHGFYPR. + 3y4 20.2 338.175 639.325 XR2-d4D.VHHGFYPR. + 3y5 18.2 338.175 578.283 XR2-d4D.VHHGFYPR. + 3b5 16.2 506.759 776.384 XR2-d4D.VHHGFYPR. + 2y6 25.8 528.772 235.108 XR3-d5D.AYDVTNFVK. + 2b2 22.9 528.772 350.135 XR3-d5D.AYDVTNFVK. + 2b3 22.9 528.772 608.34 XR3-d5D.AYDVTNFVK. + 2y5 22.9 528.772 707.409 XR3-d5D.AYDVTNFVK. + 2y6 22.9 528.772 822.436 XR3-d5D.AYDVTNFVK. + 2y7 22.9 401.227 243.13 XR3-d5D.ELVIGDR. + 2b2 18.7 401.227 342.2 XR3-d5D.ELVIGDR. + 2b3 18.7 401.227 460.251 XR3-d5D.ELVIGDR. + 2y4 18.7 401.227 559.32 XR3-d5D.ELVIGDR. + 2y5 18.7 401.227 672.4 XR3-d5D.ELVIGDR. + 2y6 18.7 388.719 216.098 XR4-d5E.SQPFGLK. + 2b2 18.9 388.719 260.197 XR4-d5E.SQPFGLK. + 2y2 26.9 338.719 317.218 XR4-d5E.SQPFGLK. + 2y3 26.9 388.719 464.287 XR4-d5E.SQPFGLK. + 2y4 26.9 388.719 561.34 XR4-d5E.SQPFGLK. + 2y5 18.9 631.797 729.38 XRS-d12D.GSSSNTEQEVPK. + 2y6 32 631.797 830.425 XR5-d12D.GSSSNTEQEVPK. + 2y7 30 631.797 944.468 XR5-d12D.GSSSNTEQEVPK. + 2y8 28 631.797 1031.5 XR5-d12D.GSSSNTEQEVPK. + 2y9 32 631.797 1019.428 XR5-d12D.GSSSNTEQEVPK. + 2b10 28 623.796 475.251 XR5-d12D.NINNC[CAM]GVGAAEK. + 2y5 29.6 623.796 631.341 XR5-d12D.NINNC[CAM]GVGAAEK. + 2y7 29.6 623.796 791.372 XR5-d12D.NINNC[CAM]GVGAAEK. + 2y8 29.6 623.796 905.415 XRS-d12D.NINNC[CAM]GVGAAEK. + 2y9 29.6 623.796 1019.458 XR5-d12D.NINNC[CAM]GVGAAEK. + 2y10 29.6 459.258 260.197 XR6-d6E.GQDPFLLK. + 2y2 28.5 459.258 373.281 XR6-d6E.GQDPFLLK. + 2y3 27.5 459.258 520.349 XR6-d6E.GQDPFLLK. + 2y4 27.5 459.258 617.402 XR6-d6E.GQDPFLLK. + 2y5 21.5 459.258 732.429 XR6-d6E.GQDPFLLK. + 2y6 19.5 577.834 234.145 XR6-d6E.VIWIFYVSK. + 2y2 33.3 577.834 496.277 XR6-d6E.VIWIFYVSK. + 2y4 33.3 577.834 643.345 XR6-d6E.VIWIFYVSK. + 2y5 29.3 577.834 756.429 XR6-d6E.VIWIFYVSK. + 2y6 27.3 577.34 942.508 XR6-d6E.VIWIFYVSK. + 2y7 25.3 361.217 400.735 XR7-d6D.ALPSRPAEIK. + 3y7++ 21.3 361.217 449.261 XR7-d6D.ALPSRPAEIK. + 3y8++ 15.3 361.217 557.329 XR7-d6D.ALPSRPAEIK. + 3y5 21.3 541.322 822.447 XR7-d6D.ALPSRPAEIK. + 2b8 29.5 541.322 897.515 XR7-d6D.ALPSRPAEIK. + 2y8 29.5 508.767 474.292 XR7-d6D.DASTAPVDLK. + 2y4 29.9 508.767 571.345 XR7-d6D.DASTAPVDLK. + 2y5 23.9 508.767 642.382 XR7-d6D.DASTAPVDLK. + 2y6 23.9 508.767 743.43 XR7-d6D.DASTAPVDLK. + 2y7 21.9 508.767 901.499 XR7-d6D.DASTAPVDLK. + 2y9 27.9 619.627 647.299 XR8-PAT.TEPQTPQEWIDDLER. + 3y5 29.7 619.627 650.812 XR8-PAT.TEPQTPQEWIDDLER. + 3y10++ 27.7 619.627 813.892 XR8-PAT.TEPQTPQEWIDDLER. + 3y12++ 25.7 928.937 946.463 XR8-PAT.TEPQTPQEWIDDLER. + 2y7 50.5 928.937 1300.617 XR8-PAT.TEPQTPQEWIDDLER. + 2y10 48.5 761.933 458.272 XR8-PAT.SVVAVIGLPNDPSVR. + 2y4 36.3 761.933 784.395 XR8-PAT.SVVAVIGLPNDPSVR. + 2y7 36.3 761.933 897.479 XR8-PAT.SVVAVIGLPNDPSVR. + 2y8 36.3 761.933 954.5 XR8-PAT.SVVAVIGLPNDPSVR. + 2y9 36.3 761.933 1067.584 XR8-PAT.SVVAVIGLPNDPSVR. + 2y10 36.3
(103) Validation of Protein Quantification by LC-MRM-MSThe MS responses (peak area) of the light peptides (analytes) were measured and plotted relative to the amount of peptide loaded onto the LC-MS system. All peptides gave a linear response over the range 0 fmol to 1,250 fmol, with the exception of the Pavsa-4D peptide LAPLV*K (aa 403-408 SEQ ID NO: 7), for which the linear range extended to 2,500 fmol as shown in
(104) TABLE-US-00016 TABLE 14 Analytical parameters for quantification of canola peptides Protein Peptide Sequence LOD LLOQ ULOQ m b R Lack1-12D GSSSNTEQEVPK 0.31 0.31 1,250 7.088e5 0.0004562 0,9980 Picpa-@3D IPFYHAR 0.61 1.22 1,250 0.0003545 0.003352 0.9974 Micpu-6D DASTAPVDLK3 0.08 0.15 1,250 0.006326 0.002506 0.9989 Pyrco-6E GQDPFLLK4 0.08 0.31 1,250 0.001400 0.007894 0.9989 Pavsa-5D AYDVTNFVKS 0.15 0.15 1,250 0.001385 0.008230 0.9988 Pyrco-5E SQPFGLK6 0.08 0.15 1,250 0.002457 0.002724 0.9996 Pavsa-4D LAPLVK7 0.08 0.31 2,500 0.001461 0.02667 0,9958 LOD, limit of detection; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; m, slope or gradient; b, y-intercept; R.sup.2, linear regression. Units in fmol loaded on-column. .sup.1GSSSNTEQEVPK: residues 16-26 of SEQ ID NO: 2: .sup.2IPFYHAR: residues 351-358 of SEQ ID NO: 1; .sup.3DASTAPVDLK: residues 30-39 of SEQ ID NO: 3; .sup.4GQDPFLLK: residues 83-90 of SEQ ID NO: 4; .sup.5AYDVTNFVK: residues 37-45 of SEQ ID NO: 5; .sup.6SQPFGLK: residues 66-72 of SEQ ID NO: 6; .sup.7LAPLVK: residues 403-408 of SEQ ID NO: 7.
(105) Levels of the 3LCPUFA Biosynthesis Pathway Enzymes in Transgenic CanolaLC-MRM-MS quantification confirmed that none of the target peptides were detected in total protein extracts from WT canola obtained at all seven sampling points at five growth stages collected from two field trial sites. Further, none of the target peptides were detected in total protein extracts in the non-seed tissues of transgenic 3LCPUFA canola obtained from the seven sampling points at five growth stages collected from two field trial sites (Table 15).
(106) TABLE-US-00017 TABLE 15 Detection of peptides in transgene proteins in canola plant parts Whole Whole Devel. Mature plant plant Root Flower Other Seed Seed Protein Peptide Sequence TG15 BTG35 TG65 TG65 TG65 TG79 TG90 Lack1-12D GSSSNTEQEVPK.sup.1 ND ND ND ND ND Picpa-@3D IPFYHAR.sup.2 ND ND ND ND ND Micpu-6D DASTAPVDLK.sup.3 ND ND ND ND ND Pyrco-6E GQDPFLLK.sup.4 ND ND ND ND ND ND Pavsa-5D AYDVTNFVK.sup.5 ND ND ND ND ND Pyrco-5E SQPFGLK.sup.6 ND ND ND ND ND ND Pavsa-4D LAPLVK.sup.7 ND ND ND ND ND .sup.1GSSSNTEQEVPK: residues 16-26 of SEQ ID NO: 2; .sup.2IPFYHAR: residues 351-358 of SEQ ID NO: 1; .sup.3DASTAPVDLK: residues 30-39 of SEQ ID NO: 3; .sup.4GQDPFLLK: residues 83-90 of SEQ ID NO: 4; .sup.5AYDVTNFVK: residues 37-45 of SEQ ID NO: 5; .sup.6SQPFGLK: residues 66-72 of SEQ ID NO: 6; .sup.7LAPLVK: residues 403-408 of SEQ ID NO: 7.
(107) All seven peptides representing the 3LCPUFA biosynthesis pathway enzymes were detected in developing or mature seeds of transgenic canola, and were quantified as shown in Table 16.
(108) TABLE-US-00018 TABLE 16 Transgenic protein quantification in developing and mature canola seed Developing seed (TG79) Mature seed (TG90) Protein Peptide Sequence Site A Site B Site A Site B Lackl-12D GSSSNTEQEVPK.sup.1 507.1 14.1 461.6 149.5 441.0 89.6 551.0 87.3 Picpa-3D IPFYHAR.sup.2 351.1 51.9 352.0 148.7 469.3 189.1 551.2 55.1 Micpu-6D DASTAPVDLK.sup.3 166.0 28.7 257.1 57.3 85.6 7.5 80.9 14.9 Pyrco-6E GQDPFLLK.sup.4 79.0 5.3 89.9 19.6 ND ND Pavsa-5D AYDVTNFVK.sup.5 131.6 34.1 136.4 65.7 129.2 31.6 155.5 41.6 Pyrco-5E SQPFGLK.sup.6 ND ND 64.1 38.7 89.7 15.7 Pavsa-4D LAPLVK.sup.7 974.6 296.6 888.7 629.1 1500 408.7 1470 313.7 The amount of peptide detected is reported in units of fmol/100 g total protein, as mean SD, n = 4. ND, not detected. .sup.1GSSSNTEQEVPK: residues 16-26 of SEQ ID NO: 2; .sup.2IPFYHAR: residues 351-358 of SEQ ID NO: 1; .sup.3DASTAPVDLK: residues 30-39 of SEQ ID NO: 3; .sup.4GQDPFLLK: residues 83-90 of SEQ ID NO: 4; .sup.5AYDVTNFVK: residues 37-45 of SEQ ID NO: 5; .sup.6SQPFGLK: residues 66-72 of SEQ ID NO: 6; .sup.7LAPLVK: residues 403-408 of SEQ ID NO: 7
(109) The Pyrco-5E and Pyrco-6E proteins revealed the lowest protein abundance in the transgenic canola (ranging from 64-90 fmol). The Pyrco-5E was below the limit of detection in developing seeds, and the Pyrco-6E protein was below the limit of detection in mature seeds. Pavsa-4D was present in the highest amount of the seven enzymes, with up to 1,500 fmol in mature seeds. Based on the molecular mass of each protein, the level of each transgenic protein was determined (on a per mg total protein basis) as shown in Table 17. Specifically, the lowest protein was Pyrco-5E at 20/mg total protein, and highest Pavsa-4D 740 ng/mg total proteins. All the detected peptides were confirmed, as shown in
(110) TABLE-US-00019 TABLE 17 Quantification of transgenic proteins in developing and mature canola seed MW Developing Seed (TG79) Mature Seed (TG90) Protein (Da) Site A Site B Site A Site B Lackl-12D 48,158 244.2 6.8 222.3 72.0 212.4 43.2 265.4 42.0 Picpa-3D 47,760 167.7 24.8 168.1 71.0 224.1 90.3 263.3 26.3 Micpu-6D 52.935 87.9 15.2 136.1 30.3 45.3 4.0 42.8 7.9 Pyrco-6E 33,078 26.1 1.8 29.7 6.5 ND ND Pavsa-5D 48,215 63.4 16.4 65.8 31.7 62.3 15.2 75.0 20.0 Pyrco-5E 31,268 ND ND 20.0 12.1 28.0 4.9 Pavsa-4D 49,307 480.5 146.2 438.2 310.2 739.5 201.5 724.7 154.7 Units are ng of transgene protein per mg total protein extracted.
(111) The seed-specific expression of the 3LCPUFA biosynthesis pathway enzymes was confirmed by examining a range of plant tissues. There was no detection of the target peptides in the non-seed tissues of the transgenic canola. The Pavsa-4D protein was the most abundant among the seven transgene products detected in developing seed or mature seed, thus was chosen as a representative protein as depicted in
(112) Protein content was detected and quantified in transgenic canola for all seven enzymes in the fatty acid biosynthetic pathway. The enzymes driving 3LCPUFA production, expressed under control of seed-specific promoters, were detected only in developing seed and mature seed, and present in low levels (20-740 ng/mg total protein). Conversely, none of the 3LCPUFA pathway enzymes were detected in other tissues of transgenic canola, regardless of the sampling time. Finally, no transgenic proteins were detected in WT canola tissues or seed.
(113) Detection of Selection Marker proteinLow level expression (below the limit of detection) of a selection marker gene (R) was confirmed in canola, as shown in
Example 4. In Vitro Stability of Pavlova Salina 4-Desaturase (Pavsa-4D
(114) This Example provides assessment of the in vitro digestibility of Pavsa-4D protein in SGF containing pepsin, in combination with a novel pepsin-trypsin assay employing state-of-the-art mass spectrometric approaches to monitor the precise degradation products. The extent of protein digestion was evaluated by the appearance and disappearance of peptic peptide products, and the disappearance of tryptic peptide products as a proxy for intact protein. Because no single method can predict the allergenicity of a protein, the allergenic potential of a protein is determined by a weight of evidence approach. Protein digestibility is one aspect of the overall allergenicity assessment that is conducted for proteins newly expressed in genetically modified crops
(115) The results of this Example show that Pavsa-4D, a recalcitrant integral membrane protein, was readily digestible in pepsin or trypsin. In a particular embodiment, the results provided herein demonstrate that upon incubation in pepsin and analyzed using LC-MS/MS, >80% of full-length Pavsa-4D protein was digested within 10 mini, and >93% of full-length Pavsa-4D protein was digested within 60 min. In another embodiment, the results provided herein demonstrate that upon incubation in pepsin and analyzed using LC-MS/MS, 99% of full-length Pavsa-4D protein was digested within 10 min, and 99.6% of full-length Pavsa-4) protein was digested within 60 min when analyzed by LC-MS/MS. Rapid digestion of the full-length protein is one of many factors that indicate transgenic protein safety.
(116) The 3LCPUFA, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid (EPA, 20:53; DPA, 22:53; and DHA, 22:63, respectively) are widely recognized for their beneficial roles in human health, particularly those related to cardiovascular and inflammatory health. EPA, DPA and DHA are sourced primarily from wild-caught fish oils and algal oils, with algae being the initial producer in the marine food web. Marine sources are under pressure, however, from increasing demand for 3LCPUFA by aquaculture, nutraceutical, and pharmaceutical applications.
(117) Additional sources of these fatty acids were produced by engineering land-based oilseed crops to convert native fatty acids to marine-type 3LCPUFA in seed oil. For example, canola is a commonly grown oilseed with 67 million metric tons (MMT) of rapeseed produced globally in 2015/16, and transgenic canola (Brassica napus) lines that produce significant amounts of 3LCPUFA, including DHA, in seed oil have been developed. As noted above, seven fatty acid desaturases and elongases were introduced into canola in a single expression vector to provide the synthesis pathway for the conversion of oleic acid (OA) to DHA. See, e.g., WO 2010/057246.
(118) Briefly, the 4-desaturase gene used in the transgenic 3LCPUFA canola was cloned from alga P. salina, codon optimized (see, e.g., WO 2010/057246), fused with a His-tag and a PreScission Protease cleavage site (SLEVLFQGP) (SEQ ID NO: 12) (GE Healthcare, Parramatta, AU), cloned into baculovirus pFastBac vector (Invitrogen, Germany), expressed in the Sf9 insect cell line, and then purified as follows: about 100 mg of insect cell pellet expressing His-Pavsa-4D was resuspended in 500 L of lysis buffer (1 phosphate buffer saline (PBS) with imidazole, DTT and PMSF). The final lysis buffer contained 140 mM NaCl, 27 mM KCl, 10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 20 mM imidazole, 10 mM DTT, 1 mM PMSF). The cells were sonicated using a Branson Probe Sonicator (Emerson Elec. Co., St. Louis, Mo., US) and centrifuged at 21,700g for 30 min at 4 C. The pellet protein and leftover protein in supernatant were assessed by SDS-PAGE and western blot analysis using a mouse anti-His-tag antibody (1:1000 dilution). The proteins were stored at 80 C. freezer until assay time.
(119) Digestibility assays employed two enzymes: trypsin and pepsin. Trypsin is a serine protease that is found in the digestive system. Trypsin cleaves polypeptide chains at the carboxyl side of the basic amino acids lysine (K) or arginine (R), but its cleavage is hindered by the presence of proline as the preceding amino acid (P1 position,
(120) Two test systems, pepsin digestion (representing simulated gastric fluid (SGF)) and a combined pepsin-trypsin digestion, were utilized independently to test the stability of the His-Pavsa-4D protein. SGF contained the proteolytic enzyme pepsin in a buffer adjusted to an acidic pH 1.2, using a highly purified form of pepsin. The SGF was formulated so that an enzyme:protein ratio of 3:1 would be present in the digestion reactions. The digestion of the Pavsa-4D protein was monitored by LC-MS/MS. The pepsin digestibility assay protocol described herein references the protocol standardized by the International Life Sciences Institute (ILS) in a multi-laboratory test and the results demonstrated that the in vitro pepsin digestion assay is reproducible when a common protocol is followed. Thomas et al., 39 Reg. Tox. Pharm. 87 (2004). Sequencing grade porcine trypsin and a highly purified form of pepsin (Catalog #V195A; specific activity >2,500 units/mg) were purchased from Promega (Madison, Wis., US). Mouse anti-His antibody (Catalog #A7058) was purchased from Sigma-Aldrich (Sydney, AU).
(121) SGF was represented by the proteolytic enzyme pepsin in a buffer adjusted to an acidic pH 1.2. The digestion was performed for 5, 10, 15, 30, and 60 min, with 0 min (no pepsin added) as the control, each with five replicates. Because of filtering and washing five replicates after pepsin digestion, the earliest practical time point was 5 min from the addition of pepsin. The increased abundance of targeted peptic peptides was used as indicator of the protein digestibility. Additionally, the SGF digestion at the time points as above was followed by 16 hr digestion with trypsin, designated as the combined pepsin-trypsin digestion. The relative abundance of tryptic peptides compared to the abundance of peptides in no pepsin (0 min) followed by trypsin digestion provided an indicator of the protein digestibility.
(122) For pepsin digestion, thirty g of protein (30 L, n=30 comprising five replicate digestions and six time-points) were applied to a 10 kDa molecular weight cut-off filter (Millipore, Australia), and washed twice with 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was exchanged using 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) by two consecutive wash/centrifugation steps. The pH was lowered by two consecutive wash/centrifugation steps with acidified 50 mM (NH.sub.4)HCO.sub.3 (pH 1.2). Numerous acids may be used to acidify buffer and achieve a low pH (i.e., pH1.2 to pH3.0) in which pepsin is active, such as HCl, acetic acid, or citric acid. The 10 kDa filters were transferred to fresh centrifuge tubes, and 90 g pepsin (150 L, 0.6 g/mL in acidified 50 mM (NH.sub.4)HCO.sub.3, pH 1.2) added to obtain an enzyme:protein ratio of 3:1. The replicate tubes were incubated at 37 C. for five time-points (5, 10, 15, 30, 60 min). Pepsin was not applied to the 0 time-point, which served as an experimental control for acid hydrolysis. The digestion was stopped by the addition of 200 L of 50 mM (NH.sub.4)HCO.sub.3, pH 8.0, which irreversibly inactivated the pepsin. The 10 kDa filters were immediately centrifuged (20,800g, 15 min) and the filtrates containing digested peptides were collected. The filters were washed twice with 200 L of 50 mM (NH.sub.4)HCO.sub.3, pH 8.0, and the filtrates combined and lyophilized, then stored at 80 C. until further analysis. For LC-MS, the peptic peptides were resuspended in 12 L of 1% formic acid and run on the QTRAP 6500+ LC-MS system and quantified.
(123) For the dual pepsin-trypsin digestion, 10 kDa filters from each time point were transferred to fresh centrifuge tubes and the residual protein was reduced with 200 l of 50 mM DTT, 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, on mixer at 600 rpm for 45 min prior to centrifugation (20,800g, 15 min). The protein was alkylated with 200 L of 50 mM IAM, 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, in the dark for 20 min prior to centrifugation (20,800g, 15 min). The 10 kDa filters were transferred to fresh centrifuge tubes, and 2 g trypsin (200 L, 0.01 g/mL in 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, and 1 mM CaCl.sub.2) was added to obtain an enzyme:protein ratio of 1.15. Replicate tubes were incubated at 37 C. for 16 hr. After incubation, the filters were centrifuged (20,800g, 15 min) and the filtrates containing digested peptides were collected. The filters were washed twice with 200 L of 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, and the filtrates combined, lyophilized, and stored at 80 C. until further analysis. For LC-MS, the tryptic peptides were resuspended in 12 L of 1% formic acid and run on a QTRAP 6500+ LC-MS and quantified.
(124) For 60 min pepsin digestion, His-Pavsa-4D protein was diluted in UA buffer to 1.3 g/L. An aliquot of the protein extract (equivalent to 200 g) was subjected to filter-assisted sample preparation (FASP). See Wisniewski et al., 2009. The protein extract was applied to a 10 kDa MWCO filter (Millipore), washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was exchanged using 50 mM (NH.sub.4)HCO.sub.3, pH 8.0, by two consecutive wash/centrifugation steps. The pH was adjusted with acidified 50 mM (NH.sub.4)HCO.sub.3 (pH 1.2) by two consecutive wash/centrifugation steps. The 10 kDa filters were transferred to fresh centrifuge tubes and 600 g pepsin (200 L, 3 g/L in 50 mM acidified (NH.sub.4)HCO.sub.3, pH 1.2) was added to obtain an enzyme:protein ratio of 3:1. The filters were incubated with the pepsin for 60 min at 37 C., then transferred to clean tubes. The filtrates (containing the digested peptides) were collected following centrifugation (20,800g, 10 min). The filters were washed with 200 L of 100 mM (NH.sub.4)HCO.sub.3 and the filtrates combined and lyophilized, then stored at 20 C. until further analysis. For LC-MS/MS, the resultant peptides were reconstituted in 12.5 L of 1% formic acid and a 10 L aliquot analyzed by LC-MS/MS.
(125) For trypsin digestion, the His-Pavsa-4D protein was diluted in UA buffer (8 M urea, 0.1 M Tris-HCl, pH 8.5) to 1.3 g/L. The protein was reduced by addition of 100 mM DTT with incubation on a shaker for 50 min at room temp. An aliquot of the protein extract (equivalent to 300 g) was subjected to F ASP. Wisniewski et al., 2009. The protein extract was applied to a 10 kDa MWCO filter, washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was then exchanged using 50 mM (NH.sub.4)HCO.sub.3, pH 8.0, by two consecutive wash/centrifugation steps. Then, 30 L sequencing grade porcine trypsin, at a concentration of 1 g/L in 100 mM (NH.sub.4)HCO.sub.3, was added to the protein on the 10 kDa filter and incubated for 16 hr at 37 C. in a wet chamber. Each filter was then transferred to a fresh centrifuge tube, and the filtrate containing the digested peptides collected following centrifugation (20,800g, 10 min). Filters were washed with 200 L of 100 mM (NH.sub.4)HCO.sub.3, and the filtrates combined and lyophilized. The tryptic peptides were subsequently resuspended in 30 L. of 1% formic acid (FA) and a 12 L aliquot analyzed by LC-MS/MS.
(126) Proteolytically digested (either pepsin or trypsin) proteins were analyzed with chromatographic separation (2%/min linear gradient from 2%-40% acetonitrile) using a nano HPLC system (Shimadzu Sci., Rydalmere, AU) coupled directly to a TripleTOF 5600 MS (AB S
(127) Either 5 L of native peptic peptides (Table 18) or reduced and alkylated tryptic peptides (Table 19) were chromatographically separated on a Nexera UHPLC (Shimadzu) and analyzed on a QTRAP 6500+ mass spectrometer (AB S
(128) For the tryptic data, peptide summaries generated by ProteinPilot software were used to select peptides that yielded intense peaks and were fully tryptic, i.e., no unusual or missed cleavages. For the pepsin data, peptide summaries generated by ProteinPilot were used to select peptides that (a) yielded intense peaks, (b) were consistently observed in the replicate digests, and (c) were present after 30 min and 60 min incubation with pepsin. As pepsin is non-specific, many of these peptide products were overlapping or contained missed cleavages. MRM transitions (Tables 18 and 19) were determined for each peptide where the precursor ion (Q1) m/z and the fragment ion (Q3) m/z values were determined from the data collected in the discovery experiments. Three transitions were used per peptide (with eight peptides from Pavsa-4D), wherein the peak area of the three MRM transitions were summed.
(129) Allergenic reactions require that a protein or protein fragment simultaneously bind to two IgE molecules in order to induce mast cell degranulation (Lack et al, 2002). This IgE binding places theoretical limits on the peptide size of between 1500 and 3500 Da. The complete digestion of a protein by a single enzyme is difficult to judge, especially when employing a non-specific enzyme such as pepsin. Although it is possible to judge the disappearance of the intact protein on a gel or by western blotting techniques, the protein may be hydrolyzed once (cleaved at a single site), or multiple times, often yielding small and overlapping fragments. Gel analysis using various staining or antibody techniques can typically detect peptides larger than 3,000 Da. Solely employing gel analysis in order to judge the completeness of digestion requires a high level of purity. When employing antibodies, the hydrolysis of a protein by a proteolytic enzyme may result in cleavage of the epitope, thus rendering antibody-based detection methods unsuitable. Likewise, cleavage of a protein at a single site may yield two protein fragments in which only one fragment contains the epitope while the other fragment does not. In that instance, large protein fragments may evade detection.
(130) By using LC-MS/MS analysis, the peptide products resulting from both pepsin and trypsin digestions can be determined qualitatively and quantitatively. LC-MS analysis can simultaneously monitor peptides spanning the entire protein length that are generated by proteolytic digestion. The combined pepsin trypsin approach to analyze digestibility, as in this Example, mimics the typical mammalian digestive system that exposes food proteins to both pepsin (stomach) and trypsin (intestine) enzymes in transit through the gut.
(131) The concentration of total Pavsa-4D protein extracted was estimated at 5.7 mg/mL, as total protein from the precipitated pellet and supernatant were assessed by SDS-PAGE (
(132) As depicted in
(133) Trypsin is comparatively specific, and digestion results in cleavage at Lys (K) and Arg (R) resulting in thirty-seven possible Pavsa-4D peptide fragments, of which twenty-two were in the mass range suited to LC-MS/MS analysis. See
(134) To assess the digestibility of the His-Pavsa-4D protein, a targeted LC-MS/MS method was developed based on the use of multiple reaction monitoring (MRM) mass spectrometry (MS). See Lange et al., 4 Mol. Syst. Biol. 222 (2008). Both the appearance and the increase of the peptic peptides during the time course of pepsin digestion were used as the evidence of the protein digestibility. Moreover, the rapid decline of the tryptic peptides subsequent to pepsin digestion served as confirmation of the protein digestibility.
(135) In order to select peptides to quantify by this method, the digestion products resulting from both pepsin and trypsin digestion were characterized. Peptides that were identified with 95% confidence and that yielded intense signals in the MS were selected for relative quantification. Eight peptides that spanned the length of the His-Pavsa-4D protein were selected from the digestion of the His-Pavsa-4D protein, and are summarized in Table 18 and Table 19.
(136) TABLE-US-00020 TABLE18 PeptideMRMtransitionsforPavsa-4Dpepsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE FTRKDVADRPDL 2.97 478.256 3+ 615.310 y5+ 27.0 (aa25-36,SEQIDNO:7) 686.347 y6+ 27.0 785.415 y7+ 27.0 TRKDVADRPDL 2.20 429.234 3+ 501.278 b4+ 24.6 (aa26-36,SEQIDNO:7) 615.310 y5+ 24.6 686.347 y6+ 24.6 FRSEHPGGAHF 2.19 414.532 3+ 657.310 b5+ 19.9 (aa49-59,SEQIDNO:7) 488.225 y5+ 23.9 585.278 y6+ 19.9 YLLPGETM 5.99 462.231 2+ 534.223 y5+ 21.6 (aa249-256,SEQIDNO:7) 673.357 b6+ 21.6 774.403 b7+ 21.6 WRWEGEPISKL 5.70 467.582 3+ 557.366 y5+ 20.4 (aa272-282,SEQIDNO:7) 715.331 b5+ 20.4 844.374 b6+ 20.4 LKRQAETSSNVGGPLL 4.19 557.313 3+ 664.850 b13++ 24.8 (aa361-376,SEQIDNO:7) 769.918 b15++ 24.8 756.425 y8+ 24.8 PRLAPLVKAEL 5.32 402.924 3+ 438.282 b4+ 19.3 (aa401-411,SEQIDNO:7) 559.345 y5+ 17.3 648.419 b6+ 17.3 APLVKAEL 4.51 420.763 2+ 460.277 y4+ 25.6 (aa404-411,SEQIDNO:7) 559.345 y5+ 21.6 672.429 y6+ 19.6 Pavsa-4Dsequence:.sup.b (SEQIDNO:7) MPPSAAKQMGASTGVHAGVTDSSAFTRKDVADRPDLTIVGDSVYDAKAFRSEHPGGAHFVSLFGGRDAT EAFMEYHRRAWPKSRMSRFHVGSLASTEEPVAADEGYLQLCARIAKMVPSVSSGFAPASYWVKAGLILG SAIALEAYMLYAGKRLLPSIVLGWLFALIGLNIQHDANHGALSKSASVNLALGLCQDWIGGSMILWLQE HVVMHHLHTNDVDKDPDQKAHGALRLKPTDAWSPMHWLQHLYLLPGETMYAFKLLELDISELVMWRWEG EPISKLAGYLFMPSLLLKLTEWARFVALPLYLAPSVHTAVCIAATVMTGSFYLAFFFFISHNFEGVASV GPDGSITSMTRGASFLKRQAETSSNVGGPLLATLNGGLNYQIEHHLFPRVHHGFYPRLAPLVKAELEAR GIEYKHYPTIWSNLASTIRHMYALGRRPRSKAE RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. .sup.bPavsa-4D sequence with mapped peptic peptides (underlined). For pepsin, different cleavage variants were observed owing to the incomplete digestion and these peptides have been differentiated by single or double underline.
(137) TABLE-US-00021 TABLE19 PeptideMRMtransitionsforPavsa-4Dtrypsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE QMGASTGVHAGVTDSSAFTR 4.00 660.647 3+ 884.411 y8+ 33.1 (aa8-27,SEQIDNO:7) 1040.501 y10+ 33.1 1111.538 y11+ 33.1 DVADRPDLTIVGDSVYDAK 5.70 683.676 3+ 953.457 y9+ 34.8 (aa29-47,SEQIDNO:7) 854.389 y8+ 34.8 797.368 y7+ 34.8 SEHPGGAHFVSLFGGR 4.95 827.908 2+ 1301.675 y13+ 38.7 (aa51-66,SEQIDNO:7) 1019.542 y9+ 38.7 882.483 y8+ 38.7 FHVGSLASTEEPVAADEGYLQLC*AR 6.22 907.438 3+ 1109.541 y9+ 47.0 (aa88-112,SEQIDNO:7) 980.498 y8+ 47.0 923.477 y7+ 47.0 DATEAFMEYHR 4.27 457.200 3+ 475.242 y3+ 24.1 (aa67-77,SEQIDNO:7) 604.284 y4+ 24.1 735.325 y5+ 24.1 VHHGFYPR 2.07 506.759 2+ 776.384 y6+ 27.1 (aa395-402,SEQIDNO:7) 639.325 y5+ 27.1 582.303 y4+ 27.1 LAPLVK 3.72 320.720 2+ 456.319 y4+ 21.0 (aa403-408,SEQIDNO:7) 527.356 y5+ 21.0 359.266 y3+ 21.0 HMYALGR 2.77 424.200 2+ 424.216 y7++ 26.2 (aa434-440,SEQIDNO:7) 579.326 y5+ 26.2 710.366 y6+ 26.2 Pavsa-4Dsequence:.sup.b (SEQIDNO:7) MPPSAAKQMGASTGVHAGVTDSSAFTRKDVADRPDLTIVGDSVYDAKAFRSEHPGGAHFVSLFGGRDAT EAFMEYHRRAWPKSRMSRFHVGSLASTEEPVAADEGYLQLCARIAKMVPSVSSGFAPASYWVKAGLILG SAIALEAYMLYAGKRLLPSIVIGWLFALIGLNIQHDANHGALSKSASVNLALGLCQDWIGGSMILWLQE HVVMHHLHTNDVDKDPDQKAHGALRLKPTDAWSPMHWLQHLYLLPGETMYAFKLLFLDISELVMWRWEG EPISKLAGYLFMPSLLLKLTFWARFVALPLYLAPSVHTAVCIAATVMTGSFYLAFFFFISHNFEGVASV GPDGSITSMTRGASFLKRQAETSSNVGGPLLATLNGGLNYQIEHHLFPRVHHGFYPRLAPLVKAELEAR GIEYKHYPTIWSNLASTLRHMYALGRRPRSKAE RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. .sup.bPavsa-4D sequence with mapped tryptic peptides (bold, underlined). For trypsin, all peptides selected were fully tryptic, i.e., contained no missed cleavages. As some of the peptides were adjacent in the sequence, these have been differentiated by single or double underline.
(138) Digestibility of His-Pavsa-4D in SGF was assessed by LC-MRM-MS method as described above. Characterization and quantification of the targeted peptic peptides showed the rapid degradation of His-Pavsa-4D. Pepsin digestion data shown in
(139) Four of the peptides characterized and quantified after pepsin digestion of His-Pavsa-4D were cleavage variants (
(140) The rapid degradation of the His-Pavsa-4D protein, demonstrated by the rapid increase of peptic peptides, was further demonstrated by rapid decline of tryptic peptides in trypsin digestion after pepsin digestion (combined pepsin-trypsin digestion). The tryptic peptides monitored after the pepsin digest show a rapid decline in the first 5-10 min and then a further decline over the remainder of the 60 min duration experiment (
(141) TABLE-US-00022 TABLE 20 Percentage of each tryptic peptide remaining during pepsin time course Time (min) Peptide Sequence 5 10 15 30 60 QMGASTGVHAGVTDSSAFTR.sup.1 46.9 14.8 10.4 10.2 5.8 DVADRPDLTIVGDSVYDAK.sup.2 28.6 6.2 5.0 3.6 3.0 SEHPGGAHFVSLFGGR.sup.3 12.3 1.0 1.2 0.6 0.4 FHVGSLASTEEPV- 23.2 2.8 1.5 1.2 0.4 AADEGYLOLC.sup.[CAM]AR.sup.4 DATEAFMEYHR.sup.5 34.2 4.8 3.2 1.8 0.9 VHHGFYPR.sup.6 51.2 18.6 12.4 11.3 5.1 LAPLVK.sup.7 47.4 16.3 11.4 12.1 6.7 HMYALGR.sup.8 45.1 8.2 7.7 5.4 3.0 .sup.1Residues (aa) 8-27, SEQ ID NO: 7; .sup.2aa 29-47, SEQ ID NO: 7; .sup.3aa 29-47, SEQ ID NO: 7; .sup.4SEQ ID NO: 11; .sup.5aa 67-77, SEQ ID NO: 7; .sup.6aa 395-402, SEQ ID NO: 7; .sup.7aa 403-408, SEQ ID NO: 7; .sup.8aa 434-440, SEQ ID NO: 7
(142) Although there were still some peptides remained after 60 min digestion, within as few as 10 min only 1% of the tryptic peptide SEHPGGAHFVSLFGGR (aa 29 47 of SEQ ID NO: 7) remained (Table 20), indicating that 99% of the intact protein was degraded. The existence of some tryptic peptides at low levels after 60 min only suggested that the intact protein was degraded into small peptides by pepsin, and these peptides were detectable. The tryptic peptide SEHPGGAHFVSLFGGR (aa 29 47, SEQ ID NO: 7) was reduced to 0.4% after 60 min, indicating that essentially there was no intact protein remained beyond this pepsin digestion time.
(143) Additionally, although Pavsa-4D, expressed as the His-tag fusion protein, could be analyzed by western blot using an anti-His-tag antibody, western blot analysis can only monitor the fusion region, rather than whole protein, which would be problematic when the His-tag is cleaved off, for example during SGF digestion. In addition, the anti-His-tag antibody is not suitable for quantification of the native Pavsa-4D (unfused) protein in transgenic canola. Thus, an alternative approach using LC-MRM-MS analysis was developed, which can be applied to both the quantification and stability of the target protein. The results herein demonstrate that the LC-MS approach is suitable for such applications. This method is at least as sensitive as traditional western blot, which normally detects in the ng to g range: the LC-MRM-MS approach detected Pavsa-4D levels as low as 7.8 femtomoles (injected on-column), which equates to 385 g on a protein scale. Additionally, although western blot may detect a limited number of epitopes (one or two) from the protein, the present embodiment targeted eight peptides, spanning the intact Pavsa-4D protein, thus providing a more complete understanding of the kinetics of digestion and the susceptibility of specific regions of the Pavsa-4D protein to proteolysis. Because of the filtration and washing steps after pepsin digestion with five replicates, the earliest practical time point during this particular protocol was 5 min. Nevertheless, this example enables use of LC-MRM-MS for protein digestibility analysis.
(144) For stability analysis of transgenic, recalcitrant or membrane-associated 3LCPUFA enzymes, Pavsa-4D protein was used as the representative of the three front-end desaturases engineered in the 3LCPUFA canola. Front-end desaturases introduce a double bond between an existing double bond and the carboxyl end of fatty acids. Additionally, front-end desaturases all contain a cytochrome b5-like domain at the N-terminus fused with a desaturase domain with three conserved histidine motifs required for desaturase activity. Zhou et al., 2007. The front-end desaturases, including 4-, 5-, 6- and 8-desaturases, exist in a wide range of organisms including algae, diatom, fungi, moss, bacteria and plants. Some of these front-end desaturases are also common in food or in food production. The results of this Example demonstrated that greater than 80% or 99% of the full-length Pavsa-4D protein digested within 10 min, and >93%, or 99.6% of the full-length Pavsa-4D protein was digested within 60 min of incubation in pepsin, when analyzed by LC-MS/MS. The combined pepsin-trypsin assay showed a rapid decline in the tryptic peptides that were used as a proxy for the presence of intact protein. In addition to rapid digestion of the full-length Pavsa-4D protein in SGF, Pavsa-4D protein represents a negligible portion of the total protein present in transgenic canola mature seed.
Example 5. The In Vitro Stability of Pyramimonas Cordata 5-Elongase (Pyrco-5E) Protein
(145) This Example characterizes the microalgae fatty acid elongase, Pyrco-5E, included in the engineering of transgenic canola to catalyze the elongation of EPA into DPA (20:5.sup.5,8,11,14,17.fwdarw.22:5.sup.7,10,13,16,19) This Example assesses the in vitro stability of this recalcitrant/intractable membrane-associated protein both in SGF comprising pepsin and in combination with the pepsin-trypsin assay, using MS to monitor precise degradation/digestion products. The extent of protein digestion was evaluated by the appearance and disappearance of peptic products and the disappearance of tryptic peptide products as a proxy for intact protein. By using LC-MS/MS analysis, the peptide products resulting from both pepsin and trypsin digestions could first be determined qualitatively and then subsequently a quantitative LC-MS/MS for the detection of these peptide fragments was developed. LC-MS analysis is capable of simultaneously monitoring peptides spanning the entire protein sequence that are generated by proteolytic digestion. The approach to analyze digestibility in this Example mimics the typical mammalian digestive system that exposes food proteins to both pepsin (stomach) and trypsin (intestine) enzymes in transit through the gut. The results described herein show that greater than 75% Pyrco-5E protein was digested within 5 min, and full-length Pyrco-5E protein was rapidly digested within 60 min of incubation in pepsin, producing a suite of pepsin peptide products <3,000 Da that spanned the entire length of the protein when analyzed using LC-MS/MS. The results show that this integral membrane protein was readily digestible in pepsin or trypsin. Rapid digestion of the full-length protein indicates that it is highly unlikely that Pyrco-5E will pose any safety concern to human health.
(146) A codon optimized Pyrco-15E gene was cloned from P. cordata and expressed in Sf9 cells using the approach described in Example 4. The Pyrco-5E protein was expressed in Sf9 insect cell line infected with baculovirus as a fusion protein with a ten-histidine residue (His) tag at the N-terminus of the protein (His-Pyrco-5E). Cells were grown by GeneArt (2 L expression in Sf99 cells infected with 1:100 virus dilution and harvested 48 hr post-infection) and the thawed cells were resuspended in lysis buffer (100 mL per 20 g of cell pellet) containing 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM DTT, 5 mM EDTA, 1 mM PMSF and two protease inhibitor tablets per 100 mL (Roche). The cells were lysed by sonication and the cellular debris removed by centrifugation. The resultant supernatant was centrifuged at 200,000g for 60 min at 4 C. to isolate the membrane fraction. The pellet was resuspended in 50 mL 20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM DTT, and 10 mM imidazole. To solubilize the His-Pyrco-5E from the membrane fraction 1% (w/v) FosCholine-16 (Glycon Biochemicals GmbH) was added to the mixture and incubated for 2 hr at 4 C. The mixture was then centrifuged for 60 min at 200,000g at 4 C. and 10 mL Ni-Sepharose FE (GE Healthcare, AU) was added to the supernatant and the slurry left to bind overnight at 4 C. After binding, the resin was poured into an empty column and washed with the binding buffer. The protein was eluted with an imidazole gradient. Fractions were analyzed by SDS-PAGE and western blots.
(147) Fractions containing the His-Pyrco-5E were pooled and buffer exchanged into MES buffer (20 mM MES pH 6.0, 50 mM NaCl, 10% glycerol, 5 mM DTT and 0.01% FosCholine-16) using a HiPrep 26/10 desalting column (GE Healthcare, AU). The sample was injected onto a 5 mL Hitrap SP column (GE Healthcare, AU) and eluted with a NaCl gradient. The fractions were analyzed by SDS-PAGE, and fractions containing the His-Pyrco-5E pooled and buffer exchanged using a HiPrep 26/10 column into PBS buffer containing 10% glycerol and 0.01% FosCholine-16. The fractions containing the His-Pyrco-5E were pooled and concentrated to 1.7 mg/mL, and flash-frozen in liquid nitrogen and stored at 80 C. Concentrated protein was analyzed by SDS-PAGE and western blotting using an anti-His HRP conjugated antibody (A7058, Sigma-Aldrich) (
(148) After extraction, the His-Pyrco-5E protein solution contained 1.7 mg/mL in PBS, 0.01% FosCholine-16, and 10% glycerol. An aliquot of the protein extract (equivalent to 5 g) was subjected to FASP. Wisniewski et al., 2009. The extract was applied to a 10 kDa MWCO filter (Millipore, AU), diluted to 200 L with UA buffer (8 M urea, 0.1 M Tris-HCl, pH 8.5) before centrifugation (20,800g, 15 min), and the filter washed with two 200 L, volumes of UA buffer with centrifugation (20,800g, 15 min). The protein on the filter was reduced by DTT (50 mM, 100 L) incubation at room temp for 50 min with shaking. The filter was washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). Cysteine residues were alkylated by LAM (50 mM, 100 L) incubation for 20 min at room temp in the dark, then the filter washed with two 200 L volumes of UA buffer with centrifugation (20,800g; 15 min). The buffer was exchanged by 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) in two consecutive wash/centrifugation steps. Sequencing grade porcine trypsin (Promega, Alexandria, Australia) was added (0.5 g in 200 L of 50 mM (NH.sub.4)HCO.sub.3, 1 mM CaCl.sub.2)) to the protein on the 10 kDa filters and incubated for 16 hr at 37 C. in a wet chamber. The filters were transferred to fresh centrifuge tubes and the filtrate (comprising digested peptides) collected following centrifugation (20,800g, 10 min). The filters were washed with 200 L of 100 mM (NH.sub.4)HCO.sub.3 and the filtrate combined and lyophilized. The tryptic peptides were resuspended in 50 L of 1% formic acid (FA) and 25 L was injected on the LC-MS/MS system.
(149) An aliquot of the His-Pyrco-5E protein extract (equivalent to 5 g) was subjected to FASP digestion. The protein extract was applied to a 10 kDa MWCO filter (Millipore), diluted to 200 L with UA buffer before centrifugation (20,800g, 15 min). The protein on the filter was washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was exchanged using 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) by two consecutive wash/centrifugation steps. The pH was adjusted by further washing with acidified 50 mM (NH.sub.4)HCO.sub.3 (pH 1.2) by two consecutive wash/centrifugation steps. The 10 kDa filter was transferred to a fresh centrifuge tube and 15 g pepsin (150 L, 0.1 g/L in 50 mM (NH.sub.4)HCO.sub.3, pH 1.2) was added to obtain an enzyme to protein ratio of 3:1. The filters were incubated at 37 C. for 120 min. The filtrate (containing the digested peptides) were collected following centrifugation (20,800g, 10 min). The filters were washed with 200 L of 100 mM (NH.sub.4)HCO.sub.3 and the filtrates were combined and lyophilised and stored at 20 C. until analysis. The resultant peptides were reconstituted in 50 L of 1% formic acid of which 25 L was analyzed by LC-MS/MS.
(150) Proteolytically digested (either pepsin or trypsin) His-Pyrco-5E protein (25 L) were analyzed with chromatographic separation (0.23%/min linear gradient from 2%-40% acetonitrile) using a Nexera UHPLC system (Shimadzu Sci., Rydalmere, AU) directly coupled to a TripleTOF 5600 MS (AB S
(151) For the tryptic data, peptide summaries generated by ProteinPilot were used to select peptides that yielded intense peaks and were fully tryptic, i.e., no unusual or missed cleavages. For the pepsin data, peptide summaries generated by ProteinPilot were used to select peptides that yielded intense peaks after 120 min incubation with pepsin. Because pepsin is non-specific, many of these peptide products were overlapping or contained missed cleavages. MRM transitions (Tables 21-22) were determined for each peptide where the precursor ion (Q1) m/z and the fragment ion (Q3) m/z values were determined from the data collected. Three transitions were used per peptide (with eight peptic peptides and a single tryptic peptide from His-Pyrco-5E), wherein the peak area of the three MRM transitions were summed.
(152) Two test systems, pepsin digestion (representing SGF) and a combined pepsin-trypsin digestion, were utilized independently to test the stability of the His-Pyrco-5E protein. SGF contained a highly purified form of the proteolytic enzyme pepsin in a buffer adjusted to an acidic pH 1.2. The SGF was formulated so that an enzyme:protein ratio of 3:1 would be present in the digestion reactions. The digestion of the Pyrco-5E protein was monitored by LC-MS/MS (as described herein).
(153) For pepsin digestion, aliquots of 6.7 g of protein (67 L, n=24 comprising four replicate digestions and six time points) were applied to a 10 kDa MWCO filter (Millipore) and diluted to 200 L UA buffer before centrifugation (20,800g, 15 min). The protein on the filter was washed twice with 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was exchanged using 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) by two consecutive wash/centrifugation steps. The pH was lowered by further washing with acidified 50 mM (NH.sub.4)HCO.sub.3 (pH 1.2) by two consecutive wash/centrifugation steps. The 10 kDa filters were transferred to fresh centrifuge tubes and 20 g pepsin (150 L, 0.133 g/L in acidified 50 mM (NH.sub.4)HCO.sub.3, pH 1.2) was added to obtain an enzyme:protein ratio of 3:1. The replicate tubes were incubated at 37 C. for five time-points (5, 10, 15, 30, or 60 min). Pepsin was not applied to the 0 time-point, which served as an experimental control for acid hydrolysis. The digestion was stopped by addition of 200 L of 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) which irreversibly inactivated the pepsin. The 10 kDa filters were centrifuged immediately (20,800g, 15 min) and the filtrate containing digested peptides were collected. The filters were washed twice with 200 L of 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) and the filtrates were combined and lyophilized and stored in a 80 C. freezer until analyzed. The peptic peptides were resuspended in 50 L of 1% formic acid, and then a 20 L aliquot was run on the QTRAP 6500+ LC-MS system and quantified.
(154) For trypsin digestion, the 10 kDa filters were transferred to fresh centrifuge tubes and the residual protein reduced with 200 L of 50 mM DTT, 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, on a mixer at 600 rpm for 45 min prior to centrifugation (20,800g, 15 min). The protein was alkylated with 200 L of 50 mM IAM, 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, in the dark for 20 min prior to centrifugation (20,800g, 15 min). The 10 kDa filters were transferred to fresh centrifuge tubes and 0.5 g trypsin (200 L, 2.5 ng/L in 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, and 1 mM CaCl.sub.2)) was added to obtain an enzyme to protein ratio of 1:15. The replicate tubes were incubated at 37 C. for 16 hr. The filters were centrifuged (20,800g, 15 min) and the filtrates containing digested peptides were collected. The filters were washed twice with 200 l of 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, and the filtrates were combined and lyophilized and stored in a 80 C. freezer until analyzed. The tryptic peptides were resuspended in 50 L of 1% formic acid and 20 L aliquots were run on the QTRAP 6500+ LC-MS and quantified.
(155) Either 20 L of native peptic peptides (Table 21) or reduced and alkylated tryptic peptides (Table 22) were chromatographically separated on a Nexera UHPLC and analyzed on a QTRAP 6500mass spectrometer. Quantification was achieved using scheduled MRM scanning experiments using a 60 sec-detection window for each MRM transition and a 0.5 sec cycle-time. Peaks were integrated using MultiQuant v3.0, in which all three transitions were required to co-elute at the same retention time (RT, min) with a signal-to-noise (S/N)>3 for detection and a S/N>5 for quantification. The graphs showing digestibility of the Pyrco-5E protein were generated in Graphpad Prism v6 software.
(156) For the dual pepsin, pepsin-trypsin assay, SGF was represented by the proteolytic enzyme pepsin in a buffer adjusted to an acidic pH 1.2. The digestion was performed for 5, 10, 15, 30 and 60 min, with 0 min (no pepsin added) as the control, each with five replicates. The increased abundance of targeted peptic peptides was used as indicator of the protein digestibility. The SGF digestion was extended by collecting samples of the pepsin digestion at the same time points, followed by 16 hr digestion with trypsin, designated as combined pepsin-trypsin digestion. The relative abundance of tryptic peptides compared to the abundance of peptides in no pepsin digestion (0 min) followed by trypsin digestion was used as indicator of the protein digestibility.
(157) The total protein extracted was estimated to be 1.7 mg/mL. The total protein from purification was assessed by SDS-PAGE (
(158) The protein was identified/characterized by LC-MS/MS analysis. Five main bands identified in the gel and western blot were excised and subjected to proteolytic digestion with trypsin. All five of the bands were identified with >99% confidence as containing His-Pyrco-5E (w/ or w/o minor contaminating proteins). The higher MW bands may be due to oligomerization (dimer, trimer, hexamer) or protein-protein interactions.
(159) Because of the difficulty of expressing and purifying membrane proteins, in general, in prokaryotic and eukaryotic systems, affinity tags like the histidine tag selected here, are commonly used. The insect cell/baculovirus system was selected because it has been used widely for expression of membrane proteins, although the yields of expressed protein is many folds less than the yields of expressed protein from systems such as E. coli.
(160) Pepsin is a relatively non-specific enzyme and its use results in cleavage at Phe (F), Tyr (Y), Trp (W) and Leu (L) resulting in hundreds of possible peptide fragments wherein missed cleavages are commonly observed. In silico analysis of the Pyrco-5E protein with pepsin digestion suggested the theoretical pepsin cleavage map shown in
(161) Trypsin is a relatively specific enzyme and its use results in cleavage at Lys (K) and Arg (R) resulting in twenty-two possible peptide fragments, of which eight were in the mass range suited to LC-MS/MS analysis. See
(162) The peptide-spectrum match w as manually verified by de novo peptide sequencing. The presence of six from six possible y-ions and additionally two b-ions confirmed the peptide as confidently identified (
(163) To assess the digestibility of the His-Pyrco-5E protein, a targeted LC-MS/MS method based on the use of multiple reaction monitoring (NRM), mass spectrometry (MS) was developed. The appearance and the increase of the peptic peptides during the time course of pepsin digestion were used as the evidence of the protein digestibility. Moreover, the rapid decline of the tryptic peptides after the pepsin digestion served as confirmation of the protein digestibility. In order to select peptides to quantify in this method, the digestion products resulting from both pepsin and trypsin digestion were characterized. Pepsin-derived peptides that were identified with 95% confidence and that yielded intense signals in the MS were selected for relative quantification. The eight peptides that were selected from the pepsin digestion of the H-is-Pyrco-5E protein and the single tryptic peptide are summarized in Tables 21-22. The selected pepsin-derived peptides spanned the length of the protein.
(164) TABLE-US-00023 TABLE21 PeptideMRMtransitionsforPyrco-5Epepsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE NVANPDIPASEKVPAYF 5.92 916.460 2+ 1108.560 y10+ 43.9 (aa20-36,SEQIDNO:6) 1221.650 y11+ 43.9 1335.690 b13++ 43.9 NVANPDIPASEKVPAY 4.73 842.930 2+ 961.500 y9+ 40.3 (aa20-35,SEQIDNO:6) 1286.660 y12+ 40.3 724.360 b7+ 40.3 GKRIMQNRSQPFGLKNAML 4.49 548.050 4+ 600.320 b10++ 25.4 (aa58-76,SEQIDNO:6) 807.440 b14++ 25.4 746.420 y7+ 25.4 GKRIMQNRSQPFGLKNAM 3.64 519.770 4+ 600.320 b10++ 24.0 (aa58-77,SEQIDNO:6) 633.330 y6+ 24.0 586.350 b5+ 24.0 LFVTSHRAQGLKV 3.89 485.950 3+ 548.810 y10++ 21.3 (aa92-104,SEQIDNO:6) 598.340 y11++ 21.3 671.880 y12++ 21.3 WGNIPDM 5.63 416.680 2+ 471.240 b4+ 19.4 (aa105-111,SEQIDNO:6) 683.310 b6+ 19.4 362.130 y3+ 19.4 LHIYHHTLL 3.99 573.824 2+ 896.490 y7+ 27.1 (aa151-159,SEQIDNO:6) 382.880 3+ 451.730 b7++ 16.4 382.880 3+ 508.270 b8++ 16.4 FVHVIM 5.20 373.207 2+ 384.200 b3+ 17.3 (aa184-189,SEQIDNO:6) 483.270 b4+ 17.3 596.350 b5+ 17.3 Pyrco-5Esequence.sup.b: (SEQIDNO:6) MASIAIPAALAGTLGYVTYNVANPDIPASEKVPAYFMQVEYWGPTIGTIGYLLFIYFGKRIMQNR SQPFGLKNAMLVYNFYQTFFNSYCIYLFVTSHRAQGLKVWGNIPDMTANSWGISQVIWLHYNNKY VELLDTFFMVMRKKFDQLSFLHIYHHTLLIWSWFVVMKLEPVGDCYFGSSVNTFVHVIMYSYYGL AALGVNCFWKKYITQIQMLQFCICASHSIYTAYVQNTAFWLPYLQLWVMVNMFVLFANFYRKRYK SKGAKKQ RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. .sup.bPyrco-5E sequence with mapped peptic peptides (bold, underlined). For pepsin, different cleavage variants were observed owing to the incomplete digestion and these peptides have been differentiated by single or double underline.
(165) The lack of available protein required the digestion protocol to be adjusted to a smaller scale (6.7 g load). As such, only a single tryptic peptide could be monitored (
(166) TABLE-US-00024 TABLE22 PeptideMRMtransitionsforPyrco-5Etrypsinproduct Peptide RT Q1m/z z Q3m/z Fragment CE SQPFLGK 3.84 388.719 2+ 561.340 y5+ 18.1 (residues66-72 317.200 y3+ 26.0 ofSEQIDNO:6) 260.200 y2+ 26.0 Pyrco-5Esequence.sup.b: (SEQIDNO:6) MASIAIPAALAGTLGYVTYNVANPDIPASEKVPAYFMQVEYWGPTIGTIGYLLFIYFGKRIMQNRSQPFGLK NAMLVYNFYQTFFNSYCIYLFVTSHRAQGLKVWGNIPDMTANSWGISQVIWLHYNNKYVELLDTFFMVMRKK FDQLSFLHIYHHTLLIWSWFVVMKLEPVGDCYFGSSVNTFVHVIMYSYYGLAALGVNCFWKKYITQIQMLQF CICASHSIYTAYVQNTAFWLPYLQLWVMVNMFVLFANFYRKRYKSKGAKKQ RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. .sup.bPyrco-5E sequence with mapped tryptic peptide (bold, underlined). For trypsin, the selected peptide was fully tryptic, i.e. contained no missed cleavages.
(167) Digestibility of His-Pyrco-5E in SGF was assessed by LC-MRM-MS method as described above. Characterization and quantification of the targeted peptic peptides showed the rapid degradation of His-Pyrco-5E. The pepsin digestion data has been presented in
(168) The rapid degradation of the His-Pyrco-5E protein demonstrated by the rapid liberation of peptic peptides was further confirmed by the decline of the single tryptic peptide after trypsin digestion (in the combined pepsin-trypsin digestion). Four of the peptides characterized and quantified after pepsin digestion were cleavage variants (
(169) In the case of the His-Pyrco-5E protein, only a single tryptic product could be detected owing to the small-scale (6.7 g load) digest and also due to the distribution of trypsin sites within the protein sequence resulting in few peptides amenable to LC-MS. The single peptide monitored, SQPFGLK (residues 66-72 of SEQ ID NO: 6), contained two pepsin cleavage sites (as indicated by the arrows) and it was expected that pepsin would cleave this peptide resulting in a decrease in peptide abundance over the time course of the pepsin digestion. After 5 min, the peptide peak area was noted to increase 3-fold, however, and remain relatively constant over the next 5 min before declining slowly over the next 50 min (
(170) TABLE-US-00025 TABLE 3 Percentage of each tryptic peptide remaining during pepsin time course Time (min) Peptide Sequence 5 10 15 30 60 SQPFGLK.sup.1 100.0 97.6 69.6 65.1 48.0 .sup.1SQPFGLK is aa 66-72 of SEQ ID NO: 6.
(171) Pyrco-5E is an integral membrane protein. Currently there is no functional antibody for western blot analysis available to quantify the transgenic protein content in 3LCPUFA canola, or detect the stability of Pyrco-5E as native protein. The commercially raised poly- and monoclonal antibodies by GenScript (Piscataway, N.J., US) failed to generate a specific signal towards Pyrco-5E. The antibodies were raised against the synthetic peptides predicted as potential epitopes for antigens (
(172) Although Pyrco-5E, expressed as the His-tag fusion protein, may be analyzed by western blot using the anti-His-tag antibody, such analysis could monitor only the fusion region, rather than whole protein, which remains problematic once the His-tag is cleaved off, for example, during SGF digestion. In addition, the anti-His-tag antibody is not suitable for quantification of the native Pyrco-5E (unfused) protein in 3LCPUFA canola. Thus, an alternative approach using LC-MRM-MS analysis was developed as described herein, which can be applied both to the quantification of target protein expressed and to the target protein stability assay. These results demonstrate that the LC-MS approach is suitable for such an application. This method is as sensitive as traditional western blot, which can normally detect proteins on a ng to g scale. In contrast, the LC-MRM-MS approach used demonstrated detected Pyrco-5E levels as low as 7.80 femtomoles (injected on-column), which equates to 2.44 g protein. In addition, western blot using antibodies might only detect a limited number of epitopes (one or two) from the target protein. In contrast, the methods described herein targeted eight peptides, spanning the intact protein, provides an understanding of the kinetics of digestion and the susceptibility of specific regions of the protein to proteolysis. Technical difficulty involved in the filtration and washing steps after pepsin digestion with four replicates, allowed an earliest practical time point was 5 min. Nevertheless, the results have shown the successful application of LC-MRM-MS for protein digestibility analysis.
(173) The Pyrco-5E protein belongs to the subfamily of microalgae fatty acid elongases that introduce a carbon to the carboxyl end of fatty acids. The Pyrco-5E protein presented in this Example for digestion analysis is a representative of the two microalgae fatty acid elongases, both recalcitrant, membrane-associated proteins, engineered into 3LCPUFA canola. The microalgae fatty acid elongases include 5-, 6- and 9-elongases, existing in a wide range of organisms including algae, diatoms, fungi, mosses, and bacteria. Desaturase activity has been assayed in crude extracts when the required substrates are added (Jackson et al., 252 Eur. J. Biochem. 513-19 (1998) but with DHA canola it is far more difficult because there are multiple desaturases and elongases expressed in the canola seed and the levels of the transgenic proteins in seed were very low. For example, Pyrco-5E was expressed at 409 ng per mg total protein in mature seed with as little as 2.78 mg of total protein extracted from 1 g of seed.
(174) The results of this Example demonstrated that the His-Pyrco-5E protein was rapidly digested over the time course of the experiment, with >75% cleavage of the N-terminal region achieved in <5 min. Within 60 min of pepsin incubation, a suite of pepsin products <3,000 Da were produced that spanned the entire peptide sequence. In addition to rapid digestion of the full-length His-Pyrco-5E protein in SGF, Pyrco-5E protein represented a negligible portion of the total protein present in 3LCPUFA canola mature seed. Rapid digestion and low expression levels are two of many factors that indicate the protein safety of Pyrco-5E protein.
Example 6. In Vitro Stability of Pichia Pastoris 3-/15-Desaturase (Picpa-3D) Protein
(175) This particular Example focuses on the representative yeast acyl-CoA type fatty acid desaturase of the pathway, P. pastoria 3-/15-desaturase (Picpa-3D) protein, which was used in the engineering of 3LCPUFA canola, to catalyze the desaturation of linoleic acid LA into -linoleic acid ALA (18:2.sup.,9,12.fwdarw.18:3.sup.9,12,15).
(176) This Example assesses the in vitro stability of the Pichia pastoris 3-/15-desaturase (Picpa-3D) protein in SGF comprising the proteolytic enzyme, pepsin, in combination with a novel pepsin-trypsin assay employing state-of-the-art mass spectrometric approaches to monitor the precise degradation products. The extent of protein digestion was evaluated by the appearance of peptic products and the disappearance of tryptic peptide products (as a proxy for intact protein). The Example shows that >80% digested within 5 min and 97% of the full-length Picpa-3D protein was digested within 60 min of incubation in pepsin, as determined using LC-MS/MS; or 98.7% of the full-length Picpa-3D protein was digested within 5 min and 99.5% of the full-length Picpa-3D protein was digested within 60 min of incubation in pepsin, as analyzed using LC-MS/MS; and shows that the integral membrane protein Picpa-3D was readily digestible in pepsin or trypsin.
(177) The 3-/15-desaturase gene used in DHA canola was cloned from alga P. pastoria (see, e.g., WO 2010/057246). The Picpa-3D protein was expressed in E. coli C41 strain as a fusion protein with green fluorescent protein (GFP) followed by eight histidine residues (8 His) at the N-terminus of the protein (His-GFP-Picpa-3D) and then purified. The vector contained coding sequence encoding a His-tag (His) and a PreScission protease (GE Healthcare) cleavage site (SLEVLFQGP) (SEQ ID NO: 12) fused to the codon optimized Picpa-3D gene.
(178) For protein extraction, the 3D protein-transformed E. coli C41 cells were grown to OD.sub.600 of 0.8, and protein expression induced with 0.5 mM IPTG at 37 C. for 4 hr. Cells were then spun down and resuspended in lysis buffer (150 mL per 60 g cell paste) containing 20 mM Hepes pH 7.6, 150 mM NaCl, 10% glycerol, 2 mM MgCl.sub.2, three Ultra complete protease inhibitor tablets per 150 mL (Roche), 1 mM PMSF, 1 mM DTT, and 1200 units of Benzonase (Merck Millipore). Cells were lysed using EmulsiFlexC5 cell homogenizer (Avestin) by three passes at 15,000 psi. After lysis, cellular debris was removed by centrifugation, then the supernatant was further centrifuged at 00,000g for 90 min at 4 C. to isolate the membrane fraction. The membrane pellet was resuspended in 50 mL of HNG buffer (20 mM Hepes, 150 mM NaCl, 10% glycerol, pH 7.6). To solubilize His-GFP-Picpa-3D from the membrane fraction 1% (w/v) FosCholine-16 (Glycon Biochemicals GmbH) was added and the mixture incubated for 3 hr at 4 C. The mixture was then centrifuged for 45 min at 200,000g at 4 C. and the supernatant loaded on a 5 mL HisTrap FF column (GE Healthcare, AU) in the presence of 10 mM imidazole and 1 mM DTT. The protein was eluted with an imidazole gradient. Fractions were analyzed by SDS-PAGE with western blotting. Fractions containing His-GFP-Picpa-3D fusion protein were pooled and concentrated to 2.5 mL using 100 kDa MWCO concentrators (Millipore). Concentrated sample was injected onto a Superdex 200 16/60 g gel filtration column (GE Healthcare) equilibrated in HNG buffer in the presence of 0.01% FosCholine-16 and 1 mM DTT. Fractions containing purified His-GFP-Picpa-3D protein were pooled, concentrated to 1.3 mg/mL, flash frozen in liquid nitrogen and stored at 80 C. Concentrated protein was analyzed by SDS-PAGE and western blotting using an anti-His HRP conjugated antibody (A7058, Sigma-Aldrich) (see
(179) For LC-MS/MS following pepsin digestion, His-GFP-Picpa-3D protein was diluted in LA buffer (8 M urea, 0.1 M Tris-HCl, pH 8.5) to 0.125 g/L. An aliquot of the protein extract (equivalent to 25 g) was subjected to FASP. Wisniewski et al., 2009. The protein extract was applied to a 10 kDa MWCO filter (Millipore), washed with two 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The filter-held protein was reduced with DTT (50 mM, 200 L) by incubation at room temp for 50 min with shaking. The filter was washed twice with 200 L UA buffer with centrifugation (20,800g, 15 min). Cysteine residues were then alkylated with IAM (50 mM, 100 L) with incubation for 40 min at room temp in the dark. The filter was washed twice with 200 L UA buffer and centrifugation (20,800g, 15 min). Buffer exchange used 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) and two consecutive wash/centrifugation steps. Sequencing grade porcine trypsin at a concentration of 0.01 g/L (2 g in 200 L of 50 mM (NH.sub.4)HCO.sub.3 with 1 mM CaCl.sub.2)) was added to the protein on the 10 kDa filters and incubated for 16 hr at 37 C. in a wet chamber. The filter was transferred to a fresh centrifuge tubes and the filtrate (digested peptides) was collected following centrifugation (20,800g, 10 min). The filters were washed with 200 L of 100 mM (NH.sub.4)HCO.sub.3 and the filtrates were combined and lyophilized. The tryptic peptides were resuspended in 50 L of 1% formic acid and 10 L was injected on the LC-MS/MS system.
(180) Further regarding LC-MS/MS, proteolytically digested (either pepsin or trypsin) protein were analyzed as described previously with chromatographic separation (2%/min linear gradient from 2%-40% acetonitrile) using a nano HPLC system (Shimadzu Scientific, Rydalmere, Australia) directly coupled to a TripleTOF 5600 MS (AB S
(181) For the tryptic data, peptide summaries generated by ProteinPilot were used to select peptides that yielded intense peaks and were fully tryptic, i.e. no unusual or missed cleavages. For the pepsin data, peptide summaries generated by ProteinPilot were used to select peptides that yielded intense peaks after 120 min incubation with pepsin. As pepsin is non-specific, many of these peptide products were overlapping or contained missed cleavages. MRM transitions (Tables 23-24) were determined for each peptide where the precursor ion (Q1) m/z and the fragment ion (Q3) m/z values were determined from the data collected in the discovery experiments. Three transitions were used per peptide (with eleven peptic and eight tryptic peptides from His-GFP-Picpa-3D), and the peak area of the three MRM transitions summed.
(182) Two test systems, pepsin digestion (representing simulated gastric fluid, SGF) and a combined pepsin-trypsin digestion, were utilized independently to test the stability of the His-GFP-Picpa-3D protein. SGF contained the proteolytic enzyme pepsin in a buffer adjusted to an acidic pH 1.2, using a highly purified form of pepsin. The SGF was formulated so that an enzyme:protein ratio of 3:1 would be present in the digestion reactions. The digestion of the Picpa-3) protein was monitored by LC-MS/MS (as described herein).
(183) By using LC-MS/MS analysis, the peptide products resulting from both pepsin and trypsin digestions could first be determined qualitatively and then subsequently a quantitative LC-MS/MS for the detection of these peptide fragments was developed. LC-MS analysis is capable of simultaneously monitoring peptides spanning the entire protein sequence that are generated by proteolytic digestion. The approach to analyze digestibility in this Example may mimic the typical mammalian digestive system that exposes food proteins to both pepsin (stomach) and trypsin (intestine) enzymes in transit through the gut.
(184) For pepsin digestion, 25 g of protein (58.4 L, n=30 comprising five replicate digestions and six time-points) were applied to a 10 kDa molecular weight cut-off filter (Millipore, Australia), washed twice with 200 L volumes of UA buffer with centrifugation (20,800g, 15 min). The buffer was exchanged using 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0) by two consecutive wash/centrifugation steps. The pH was adjusted by further washing with acidified 50 mM (NH.sub.4)HCO.sub.3 (pH 1.2) by two consecutive wash/centrifugation steps. A number of acids may be used to bring the pH of the digestion buffer to pH 1.5-pH 2.5, such as HCl, acetic acid, or citric acid. The 10 kDa filters were transferred to fresh centrifuge tubes and 84 g pepsin (150 L, 0.562 g/mL in acidified 50 mM (NH.sub.4)HCO.sub.3 (pH 1.2) was added to obtain an enzyme to protein ratio of 3:1. The replicate tubes were incubated at 37 C. for five time-points (5, 10, 15, 30, 60 min). Pepsin was not applied to the 0 time-point, which served as an experimental control for acid hydrolysis. The digestion was stopped by the addition of 200 L of 50 mM (NH.sub.4)HCO.sub.3 (pH 8.0), which irreversibly inactivated the enzyme. The 10 kDa filters were immediately centrifuged (20,800g, 15 min) and the filtrate containing digested peptides were collected. The filters were washed twice with 200 L of 50 mM (NH.sub.4)HCO.sub.3, pH 8.0, and the filtrates were combined and lyophilized and stored in a 80 C. freezer until further analysis. For C-MS, the peptic peptides were resuspended in 50 L of 1% formic acid, and a 3 L aliquot run on a QTRAP 6500+ LC-MS system and quantified.
(185) For trypsin digestion, the 10 kDa filters were transferred to fresh centrifuge tubes and the residual protein reduced with 200 L of 50 mM DTT, 50 mM (NH.sub.4)HCO.sub.3 (pH 8.5) on mixer at 600 rpm for 45 min prior to centrifugation (20,800g, 15 min). The protein was alkylated with 200 L of 50 mM iodoacetamide (IAM), 50 mM (NH.sub.4)HCO.sub.3, at pH 8.5, in the dark for 20 min prior to centrifugation (20,800g, 15 min). The 10 kDa filters were transferred to fresh centrifuge tubes and 2 g trypsin (200 L, 0.01 g/mL in 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, and 1 mM CaCl.sub.2)) was added to obtain an enzyme:protein ratio of 1:15. Replicate tubes were incubated at 37 C. for 16 hr. The filters were centrifuged (20,800g, 15 min) and the filtrates containing digested peptides collected. The filters were washed twice with 200 L of 50 mM (NH.sub.4)HCO.sub.3, pH 8.5, and the filtrates combined and lyophilized and stored in a 80 C. freezer until further analysis. For LC-MS, the tryptic peptides were resuspended in 50 L of 16% formic acid, and a 3 L aliquot run on a QTRAP 6500+ LC-MS system and quantified.
(186) For LC-MS/MS quantification of the digestion products, either 3 L of native peptic peptides (Table 23) or reduced and alkylated tryptic peptides (Table 24) were chromatographically separated on a Nexera UHPLC (Shimadzu) and analyzed on a QTRAP 6500+ mass spectrometer (AB S
(187) Pepsin is a protease produced in the stomach and is efficient at cleaving the peptide bonds adjacent to aromatic and hydrophobic amino acids phenylalanine, tyrosine, tryptophan, and leucine (
(188) By employing trypsin post-pepsin (see
(189) Therefore, for this digestibility assay, two enzymes were used pepsin and trypsin. Tryptic peptide products were used as a proxy for intact protein, whereby in the absence of pepsin the amount of tryptic peptide present was equated to 100% of protein being present. In the presence of pepsin (at varying time points during digestion), the level of tryptic peptides would be expected to decrease for peptides that contained a pepsin cleavage site. In this way the complete degradation of the protein was monitored.
(190) SGF was represented by the proteolytic enzyme pepsin in a buffer adjusted to acidic pH 1.2. The digestion was performed for 5, 10, 15, 30, and 60 min, with 0 min (no pepsin added) as the control, each with five replicates. Due to the difficulty involved in filtering and washing with five replicates, an early practical time point was 5 min from the addition of pepsin. The increased abundance of targeted peptic peptides was used as indicator of the protein digestibility. The SGF digestion was further extended by pepsin digestion at the same time points as above, followed by a 16 hr digestion with trypsin, designated as combined pepsin-trypsin digestion. The relative abundance of pepsin-trypsin tryptic peptides, compared with the abundance of same peptides in no-pepsin digestion (0 min, no pepsin) followed by trypsin digestion, was used as indicator of the protein digestibility.
(191) The His-GFP-Picpa-3D fusion protein ran, during electrophoresis, as a doublet with apparent molecular weights of 50 kDa and 60 kDa as determined by SDS-PAGE and western blotting. The predicted molecular weight of the His-GFP-Picpa-3D fusion construct is 76.4 kDa, however, a lower than predicted apparent molecular weight on SDS-PAGE is a common and well-documented phenomenon for membrane proteins caused by the presence and binding of detergent to the hydrophobic regions. Rath et al., 2009. In addition, the presence of two separate bands (
(192) The protein was also identified/characterized by LC-MS/MS analysis after proteolytic digestion using both trypsin and pepsin. Because of the difficulty of expressing membrane proteins in general in prokaryotic or eukaryotic systems, the strategy was to express the Picpa-3D as a. GFP fusion in E. coli with a His-tag added to aid in purification. GFP is widely used as a fusion partner for soluble expression and allowing tracking of protein expression by monitoring its fluorescence. The presence of the fusion partner (His-GFP) is unlikely to affect the proteolysis and LC-MS characterization of the His-GFP-Picpa-3D protein, nor is the presence of contaminating proteins in the mixture.
(193) As noted above, pepsin is a relatively non-specific enzyme and its use results in cleavage at Phe (F), Tyr (Y), Trp (W) and Leu (L), resulting in hundreds of possible peptide fragments wherein missed cleavages are commonly observed. In silico analysis of the native Picpa-3D protein with pepsin digestion suggested the theoretical pepsin cleavage map shown in
(194) In contrast, trypsin is a relatively specific enzyme and its use results in cleavage at Lys (K) and Arg (R) resulting in thirty-six possible peptide fragments (
(195) To assess the digestibility of the Picpa-3D protein, a targeted LC-MS/MS method based on the use of multiple reaction monitoring (MRM) (Lange et al., 2008) mass spectrometry (MS) was developed. The appearance and the increase of the peptic peptides during the time course of pepsin digestion were used as the evidence of the protein digestibility. Moreover, the rapid decline of the tryptic peptides subsequent to pepsin digestion served as confirmation of the protein digestibility.
(196) In order to select peptides to quantify in this method, the digestion products resulting from both pepsin and trypsin digestion were first characterized. Peptides that were identified with 95% confidence and that yielded intense signals in the MIS were selected for relative quantification. Eleven peptides were selected from the digestion of the H-is-GFP-Picpa-3D protein with pepsin and eight peptides for trypsin digestion are summarized in Tables 23-24. The selected peptides spanned the length of the protein.
(197) TABLE-US-00026 TABLE23 PeptideMRMtransitionsforPicpa-3Dpepsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE FKVPDYTIKDIL 6.66 484.610 3+ 601.392 y5+ 21.3 (aa42-53,SEQIDNO:1) 702.440 y6+ 21.3 587.319 b5+ 21.3 FKVPDY 4.42 384.702 2+ 394.161 y3+ 17.9 (aa42-47,SEQIDNO:1) 621.324 y5+ 17.9 587.319 b5+ 17.9 TIKDIL 4.60 351.723 2+ 488.308 y4+ 16.2 (aa48-53,SEQIDNO:1) 601.392 y5+ 16.2 571.345 b5+ 16.2 TYIPLLPNEF 8.07 603.824 3+ 619.309 y5+ 27.0 (aa88-97,SEQIDNO:1) 829.445 y7+ 27.0 701.423 b6+ 27.0 TYIPLLPNE 6.75 530.290 3+ 682.377 y6+ 23.5 (aa88-96,SEQIDNO:1) 588.339 b5+ 23.5 701.423 b6+ 23.5 TYIPLL 7.34 360.221 2+ 378.202 b3+ 16.7 (aa88-93,SEQIDNO:1) 475.255 b4+ 16.7 588.339 b5+ 16.7 IPLLPNEF 7.36 471.768 2+ 437.312 b4+ 22.1 (aa90-97,SEQIDNO:1) 648.408 b6+ 22.1 777.451 b7+ 22.1 IPLLPNE 5.74 398.234 2+ 324.228 b3+ 18.5 (aa90-96,SEQIDNO:1) 437.312 b4+ 18.5 648.408 b6+ 18.5 NATGQPYPGVSKF 4.32 683.344 3+ 894.472 y8+ 30.8 (aa221-233,SEQIDNO:1) 1079.552 y9+ 30.8 1180.600 y10+ 30.8 FKSHYWPSSPVF 5.69 494.579 3+ 560.764 b9++ 21.7 (aa234-245,SEQIDNO:1) 609.291 b10++ 21.7 658.825 b11++ 21.7 TAYKVFGF 5.89 466.748 2+ 597.340 y5+ 21.9 (aa269-276,SEQIDNO:1) 760.403 y6+ 21.9 831.440 y7+ 21.9 Picpa-3Dsequence.sup.b: (SEQIDNO:1) MSKVTVSGSEILEGSTKTVRRSGNVASFKQQKTAIDTFGNVFKVPDYTIKDILDAIPKHCYERSLVKS MSYVVRDIVAISAIAYVGL IPLL
FLRFAAWSAYVESISCFGFGIWILGHECGHSAFSNYGWVN DTVGWVLHSLVMVPYFSWKFSHAKHHKATGHMTRDMVFVPYTAEEFKEKHQVTSLHDIAEETPIYSVF ALLFQQLGGISLYLATNATGQPYPGVSKFFKSHYWPSSPVFDKKDYWYIVLSDLGILATLTSVYTAYK VFGFWPTFITWFCPWILVNHWLVFVTFLQHTDSSMPHYDAQEWTFAKGAAATIDREFGILGIIFHDII ETHVLHHYVSRIPFYHAREATECIKKVMGEHYRHTDENMWVSLWKTWRSCQFVENHDGVYMERNCNNV GVKPKDT RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. .sup.bPicpa-3D sequence with mapped peptic peptides (bold, underlined). For pepsin, different cleavage variants were observed owing to the incomplete digestion and these peptides have been differentiated by single, double or waved underline.
(198) TABLE-US-00027 TABLE24 PeptideMRMtransitionsforPicpa-3Dtrypsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE VTVSGSEILEGSTK 4.40 703.870 2+ 1020.250 y10+ 33.5 (aa4-17,SEQIDNO:1) 1107.550 y11+ 33.5 1206.620 y12+ 33.5 SGNVASFK 2.41 405.210 2+ 452.250 y4+ 18.9 (aa22-29,SEQIDNO:1) 665.360 y6+ 18.9 551.320 y5+ 18.9 TAIDTFGNVFK 5.90 606.820 2+ 927.460 y8+ 28.7 (aa33-43,SEQIDNO:1) 1040.540 y9+ 28.7 812.430 y7+ 28.7 VPDYTIK 3.42 418.230 2+ 736.390 y6+ 19.5 (aa44-50,SEQIDNO:1) 368.690 y6++ 19.5 639.330 y5+ 19.5 DILDAIPK 3.42 442.760 2+ 656.400 y6+ 20.7 (aa51-58,SEQIDNO:1) 543.310 y5+ 20.7 244.170 y2+ 20.7 EATEC.sup.[CAM]IK 1.43 425.700 2+ 549.240 y4+ 19.9 (SEQIDNO:9) 650.290 y5+ 19.9 325.650 y5++ 19.9 HTDENMWVSLWK 6.45 515.910 3+ 533.300 y4+ 24.3 (aa374-385,SEQID 632.380 y5+ 24.3 NO:1) 818.450 y6+ 24.3 SC.sup.[CAM]QFVENHDGV 5.07 630.260 3+ 772.380 y6+ 29.9 YMFR 887.410 y7+ 29.9 (SEQIDNO:10) 1024.470 y8+ 29.9 Picpa-3Dsequence:.sup.b (SEQIDNO:1) MSKVTVSGSEILEGSTKTVRRSGNVASFKQQKTAIDTFGNVFKVPDYTIKDILDAIPKHCYERSLVKSMSYV VRDIVAISAIAYVGLTYIPLLPNEFLRFAAWSAYVFSISCFGFGIWILGHECGHSAFSNYGWVNDTVGWVLH SLVMVPYFSWKFSHAKHHKATGHMTRDMVFVPYTAEEFKEKHQVTSLHDIAEETPIYSVFALLFQQLGGLSL YLATNATGQPYPGVSKFFKSHYWPSSPVFDKKDYWYIVLSDLGILATLTSVYTAYKVFGFWPTFITWFCPWI LVNHWLVFVTFLQHTDSSMPHYDAQEWTFAKGAAATIDREFGILGIIFHDIIETHVLHHYVSRIPFYHAREA TECIKKVMGEHYRHTDENMWVSLWKTWRSCQFVENHDGVYMFRNCNNVGVKPKDT RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. .sup.bPicpa-3D sequence with mapped tryptic peptides (bold, underlined). For trypsin, all peptides selected were fully tryptic, i.e., contained no missed cleavages. As some of the peptides were adjacent in the sequence, these have been differentiated by single or double underline.
(199) Digestibility of the His-GFP-Picpa-3D in SGF was assessed by LC-MRM-MS method as described herein. Characterization and quantification of the targeted peptic peptides showed the rapid degradation of the His-GFP-Picpa-3D protein. The pepsin digestion data has been presented in
(200) Five of the peptides characterized and quantified after pepsin digestion were cleavage variants (
(201) The tryptic peptides monitored after the pepsin digest show a rapid decline in the first 5 min and then a further decline over the remainder of the experiment (60 min duration). It is estimated that >97% of the protein is cleaved after 60 min on the basis of the disappearance of these eight tryptic peptides. The peptides containing multiple pepsin cleavage sites: TAIDTFGNVFK (aa 33-43, SEQ ID NO: 1), HTDENMWVSLWK (aa 374-385 of SEQ ID NO: 1), SC.sup.[CAM]QFVENHDGVYMFR (SEQ ID NO: 10) (where XX represent pepsin cleavage site) are reduced to 0.9, 1.1 and 0.3% of the undigested control (no pepsin digest) respectively. This is supported by analysis of the digested peptides on the TripleTOF 5600 LC-MS/MS, which shows that these peptides are more frequently fragmented to yield smaller fragments after 30-60 min. The tryptic peptides containing fewer sites: VTVSGSEILEGSTK (aa 4-17, SEQ ID NO: 1), SGNVASFK (aa 22-29, SEQ ID NO: 1) and VPDYTIK (aa 44-50, SEQ ID NO: 1) (with a single site) or DILDAIPK (aa 51-58, SEQ ID NO: 1) (where the lysine in position P3 is known to hinder pepsin cleavage) were reduced to 0.7, 0.5, 1.3 and 1.4% respectively. The higher percentage of EATECIK (SEQ ID NO: 9) observed (17.5% at 5 min and 2.7% at 60 min) can be explained by the absence of peptic digestion sites within this peptide sequence. Overall, it was observed that the peptides from the termini (both N- and extreme C-termini) of the protein were liberated rapidly with <6% remaining after 5 min (Table 25). Within as short as 5 min, only 1.3% of the tryptic peptide SGNVASFK (aa 22-29, SEQ ID NO: 1) remained (Table 25), indicating that 98.7% of the intact protein was degraded. The existence of some tryptic peptides at low levels after 60 min solely suggested that the intact protein was degraded into small peptides by pepsin, and these peptides were detectable. The tryptic peptide SGNVASFK (aa 22-29, SEQ ID NO: 1) was reduced to 0.56% after 60 min, indicating that essentially there was no intact protein remained beyond this pepsin digestion time.
(202) TABLE-US-00028 TABLE 25 Percentage of each tryptic peptide remaining during pepsin time course Incubation time (min) Peptide Sequence 5 10 15 30 60 VTVSGSEILEGSTK.sup.1 2.8 1.5 1.8 1.3 0.7 SGNVASFK.sup.2 1.3 1.1 1.0 1.0 0.5 TAIDTFGNVFK.sup.3 2.5 1.7 2.6 1.4 0.9 VPDYTIK.sup.4 3.8 2.7 3.6 2.3 1.3 DILDAIPK.sup.5 6.0 6.1 5.5 3.7 1.4 EATEC.sup.[CAM]IK.sup.6 17.5 17.1 18.1 9.4 2.7 HTDENMWVSLWK.sup.7 16.5 9.2 9.9 8.2 1.1 SC.sup.[CAM]QFVENHDGVYMFR.sup.8 2.0 1.3 1.1 0.9 0.3 .sup.1residues (aa) 4-17 of SEQ ID NO: 1; 2aa 22-29 of SEQ ID NO: 1; .sup.3aa 33-43 of SEQ ID NO: 1; .sup.4aa 44-50 of SEQ ID NO: 1; .sup.5aa 51-58 of SEQ ID NO: 1; .sup.6SEQ ID NO:9; .sup.7aa 374-385 of SEQ ID NO: 1; .sup.8SEQ ID NO: 10.
(203) Picpa-3D is an integral membrane protein. Until recently, there is no functional antibody for western blot analysis available to quantify the transgenic protein content in transgenic canola, or detect the stability of Picpa-3D as a native protein. The commercially raised polyclonal and monoclonal antibodies (GenScript, Piscataway, NJ, US) failed to generate a specific signal towards Picpa-3D. The antibodies were raised against the synthetic peptides predicted as potential epitopes for antigens (
(204) Although Picpa-3D expressed as the His-GFP fusion protein could be analyzed by western blot against the anti-His-tag antibody, such a western blot analysis could only monitor the fusion region, rather than whole protein, when the His-tag is cleaved off, for example after SGF digestion. In addition, the anti-His-tag antibody is not suitable for quantification of the native Picpa-3D (unfused) protein in transgenic 3LCPUFA canola. Thus, an alternative approach using LC-MRM-MS analysis was developed here, which can be applied both for the quantification of protein expressed in canola and for the stability assays. The results shown here clearly demonstrated that the IC-MS approach is suitable for such an application. This method is as sensitive as traditional western blot, which can normally detect ng to g of protein. The LC-MRM-MS approach described herein detected as little as 7.86 fmol (injected on-column) which equates to 372 g. In addition, western blot using antibodies might only detect a limited number of epitopes (one or two) from the protein. Here, eleven (peptic) and eight (tryptic) peptides were targeted, along with the intact protein, which provides an understanding of the kinetics of digestion and the susceptibility of specific regions of the protein to proteolysis. Due to the technical difficulty that was involved in filtering and washing steps after pepsin digestion with five replicates, the earliest practical time point was 5 min. Nevertheless, the results show the successful application of LC-MRM-MS for target protein digestibility analysis. For example, Picpa-3D) was expressed at 352 ng per mg of total protein in mature seed with as little as 2.78 mg of total protein extracted from 1 g of seed.
(205) The Picpa-3D protein belongs to the subfamily of yeast acyl-CoA type fatty acid desaturases that introduce a double bond between the 15-position from the carboxyl end of fatty acids. The yeast acyl-CoA type fatty acid desaturases include 12- and 3-/15-desaturases. Some of these yeast acyl-CoA type fatty acid desaturases are also common in food, animal feeds or in food production. The Picpa-3D protein was used as the representative of two yeast acyl-CoA type fatty acid desaturases (Picpa-3D and Lack1-12 desaturase) engineered into 3LCPUFA canola.
(206) The results of this Example demonstrate that the combined pepsin-trypsin assay showed a rapid decline in the tryptic peptides that were used as a proxy for the presence of intact protein. 98.7% of the full-length Picpa-3D protein was digested within 5 min, and 99.5% was digested within 60 min of incubation in pepsin. In addition to rapid digestion of Picpa-3D protein in SGF, Picpa-3D protein represents a negligible portion of the total protein present in the transgenic 3LCPUFA
Example 7. LC-MS/MS Analysis of Pavlova Salina 5-Desaturase Protein Stability
(207) assess the in vitro stability of the Pavlova salina 5-desaturase (Pavsa-5D) protein in simulated gastric fluid (SGF) comprising the proteolytic enzyme, pepsin, and in combination with a novel pepsin-trypsin assay and LC-MS/MS to monitor the precise degradation products. The extent of protein digestion was evaluated by the appearance of peptic products and the disappearance of tryptic peptide products (as a proxy for intact protein). The method in this Example demonstrated that 99.9% of the full-length Pavsa-5D protein was digested within 5 min, and six out of twelve peptides were present <0.2% after 60 min of incubation in pepsin.
(208) The 5-desaturase gene used in DHA canola was previously cloned from alga P. salina. The Pavsa-5D protein was expressed as a His-tag fusion in Sf9 insect cell line infected with recombinant baculovirus constructed using pFastBac vector (Invitrogen, DE) and then purified. The vector contained coding sequences encoding a His-tag (His10) and a PreScission protease cleavage site (SLEVLFQGP) fused to the codon optimized Pavsa-5D gene produce fusion protein His10::Pavsa-5D. The main band identified in the gel and Western Blot was excised and subjected to proteolytic digestion with trypsin. The band was identified with >99% confidence as containing His10::Pavsa-5D (with or without minor contaminating proteins). The other minor bands are likely to be insect cell proteins. The recombinant protein was also analyzed by LC-MS/MS. Protein extraction, digestions, LC-MS characterization of the protein post-trypsin digestion and post-pepsin digestion, peptide summaries, and general assays were conducted as described above. Three transitions were used per peptide (with 11 peptides from Pavsa-5D), wherein the peak area of the three MRM transitions were summed.
(209) Two test systems, pepsin digestion (representing simulated gastric fluid, SGF) and a combined pepsin-trypsin digestion, were utilized independently to test the stability of the His10::Pavsa-5D protein. SGF contained the proteolytic enzyme pepsin in a buffer adjusted to an acidic pH 1.2, using a highly purified form of pepsin. The SGF was formulated so that an enzyme:protein ratio of 3:1 would be present in the digestion reactions. The digestion of the Pavsa-5D protein was monitored by LC-MS/MS.
(210) In this study, the peptide fragments of His10::Pavsa-5D persisting after pepsin digestion for 120 min were characterized by untargeted LC-MS/MS. Protein sequence coverage obtained after pepsin digestion is depicted below, in which bold indicates peptides identified with >95%0 confidence; italics indicates peptides identified with 50-95% confidence; underline indicates peptides identified with <50% confidence; normal means residues were not detected; the wave underline is the N-terminal His-tag and protease cleavage site followed by methionine of native Pavsa-5D in the fusion protein:
(211) TABLE-US-00029 (SEQIDNO:17) MPPRDSYSYAAPPSAQLHEVDTPQEHDKK ELVIGDRAYDVTNFVKRHPGGKIIAYQVGTDATDAYKQFHVRSAKADKM LKSLPSRPVHKGYSPRRADLIADFQEFTKQLEAEGMFEPSLPHVAYRLA EVIAMHVAGAALIWHGYTFAGIAMLGVVQGRCGWLMHEGGHYSLIGNIA FDRAIQVACYGLGCGMSGAWWRNQHNKHHATPQKLQHDVDLDTLPLVAF HERIAAKVKSPAMKAWLSMQAKLFAPVTTLLVALGWQLYLHPRHMLRTK HYDELAMLGIRYGLVGYLAANYGAGYVLACYLLYVQLGAMYIFCNFAVS HTHLPVVEPNEHATWVEYAANHTTNCSPSWWCDWWMSYLNYQIEHHLYP SMPQFRHPKIAPRVKOLFEKHGLHYDVRGYFEAMADTFANLDNVAHAPE KKMQ
(212) Protein sequence coverage obtained after trypsin digestion was characterized by LC-MS/MS and is depicted below, in which bold indicates peptides identified with >95% confidence; italics indicates peptides identified with 50-95% confidence; underline indicates peptides identified with <50% confidence; normal means residues were not detected; the wave underline is the N-terminal His-tag and protease cleavage site followed by methionine of native Pavsa-5D in the fusion protein:
(213) TABLE-US-00030 (SEQIDNO:17) MPPRDSYSYAAPPSAQLHEVDTPQEHDKK ELVIGDRAYDVTNFVKRHPGGKIIAYQVGTDATDAYKQFHVRSAKADKM LKSLPSRPVHKGYSPRRADLIADFQEFTKQLEAEGMFEPSLPHVAYRLA EVIAMHVAGAALIWHGYTFAGIAMLGVVQGRCGWLMHEGGHYSLTGNIA FDRAIQVACYGLGCGMSGAWWRNOHNKHHATPQKLQHDVDLDTLPLVAF HERIAAKVKSPAMKAWLSMQAKLFAPVTTLLVALGWQLYLHPRHMLRTK HYDELAMLGIRYGLVGYLAANYGAGYVLACYLLYVQLGAMYIFCNFAVS HTHLPVVEPNEHATWVEYAANHTTNCSPSWWCDWWMSYLNYQIEHHLYP SMPQFRHPKIAPRVKOLFEKHGLHYDVRGYFEAMADTFANLDNVAHAPE KKMQ
(214) Peptides that were identified with 95% confidence and that yielded intense signals in the MS were selected for relative quantification. The 11 pepsin-derived and 12 trypsin-derived peptides that were selected from the digestion of the His10::Pavsa-5D protein are summarized in Tables 1-2. The selected peptides spanned the length of the protein. Details are shown in Tables 26 and 27
(215) TABLE-US-00031 TABLE26 PeptideMRMtransitionsforPavsa-5Dpepsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE SYAAPPSAQL 4.52 502.756 2+ 745.351 b8+ 23.6 612.335 y6+ 683.372 y7+ YAAPPSAQL 4.42 459.240 2+ 786.378 b8+ 21.5 612.335 y6+ 658.319 b7+ FVKRHPGGKIIA 2.13 661.900 2+ 1120.673 b10+ 31.4 879.494 b8+ 1007.589 b9+ VKRHPGGKIIA 1.03 588.372 2+ 860.521 b8+ 27.8 655.413 y7+ 948.573 y9+ LKSLPSRPVHKGYSPRRADL 2.69 456.260 5+ 581.820 y10++ 20.8 650.349 y11++ 517.770 y9++ QEFTKQ 1.12 390.698 2+ 634.319 b5+ 18.1 523.287 y4+ 652.330 y5+ FAPVTTL 5.85 374.715 2+ 516.281 b5+ 17.4 617.329 b6+ 530.318 y5+ YLHPRHMLRTKHYDEL 3.00 528.025 4+ 721.864 y11++ 24.4 848.440 y13++ 676.293 y5+ DVRGYF 4.30 378.687 2+ 428.225 b4+ 17.6 591.288 b5+ 542.272 y4+ DVRGY 1.65 305.150 2+ 371.203 b3+ 14.0 395.203 y3+ 494.272 y4+ DNVAHAPEKKMQ 1.22 684.340 2+ 760.402 y6+ 32.5 831.439 y7+ 968.498 y8+ DNVAHAPEKKM*Q 0.66 692.340 2+ 776.402 y6+ 32.9 847.439 y7+ 984.498 y8+ Pavsa-5Dsequence:.sup.b (SEQIDNO:5) MPPRDSYSYAAPPSAQLHEVDTPQEHDKKELVIGDRAYDVTNFVKRHPGGKIIAYQVGTDATDAYKQFHVR SAKADKMLKSLPSRPVHKGYSPRRADLIADFQEFTKQLEAEGMFEPSLPHVAYRLAEVIAMHVAGAALIWH GYTFAGIAMLGVVQGRCGWLMHEGGHYSLTGNIAFDRAIQVACYGLGCGMSGAWWRNQHNKHHATPQKLQH DVDLDTLPLVAFHERIAAKVKSPAMKAWLSMQAKLFAPVTTLLVALGWQLYLHPRHMLRTKHYDELAMLGI RYGLVGYLAANYGAGYVLACYLLYVQLGAMYIFCNFAVSHTHLPVVEPNEHATWVEYAANHTINCSPSWWC DWWMSYLNYQIEHHLYPSMPQFRHPKIAPRVKQLFEKHGLHYDVRGYFEAMADTFANLDNVAHAPEKKMQ RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. The amino acid marked with * represents modified form: oxidation (Met). .sup.bThe mature Pavsa-5D sequence with mapped peptic peptides (bold, underlined). For pepsin, different cleavage variants were observed owing to the incomplete digestion and these peptides have been differentiated by single or double underline.
(216) TABLE-US-00032 TABLE27 PeptideMRMtransitionsforPavsa-5Dtrypsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE DSYSYAAPPSAQLHEVDTPQEHDK 4.29 672.058 4+ 915.935 y16++ 31.6 964.461 y17++ 753.353 y6+ ELVIGDR 3.62 401.227 2+ 559.320 y5+ 18.7 460.251 y4+ 243.130 b2+ AYDVTNFVK 4.85 528.772 2+ 822.436 y7+ 24.9 707.409 y6+ 608.340 y5+ IIAYQVGTDATDAYK 4.80 814.912 2+ 1040.489 y10+ 38.9 941.421 y9+ 884.400 y8+ SLPSRPVHK 1.44 510.801 2+ 820.479 y7+ 24.0 723.426 y6+ 636.394 y5+ ADLIADFQEFTK 7.11 699.351 2+ 1211.631 y10+ 33.3 985.463 y8+ 799.398 y6+ QLEAEGMFEPSLPHVAYR 6.37 692.000 3+ 742.400 y6+ 31.2 1039.569 y9+ 1168.612 y10+ LQHDVDLDTLPLVAFHER 6.83 706.704 3+ 968.531 y8+ 31.9 871.478 y7+ 758.394 y6+ AWLSMQAK 4.60 467.744 2+ 677.365 y6+ 21.9 564.281 y5+ 477.249 y4+ HYDELAMLGIR 6.04 439.890 3+ 458.309 y4+ 19.1 589.340 y5+ 660.380 y6+ HGLHYDVR 2.21 498.754 2+ 859.442 y7+ 23.4 802.421 y6+ 689.337 y5+ GYFEAMADTFANLDNVAHAPEK 7.58 804.370 3+ 865.453 y8+ 36.6 980.480 y9+ 1207.600 y11+ Pavsa-5Dsequence: (SEQIDNO:5) MPPRDSYSYAAPPSAQLHEVDTPQEHDKKELVIGDRAYDVTNFVKRHPGGKIIAYQVGTDATDAYKQFH VRSAKADKMLKSLPSRPVHKGYSPRRADLIADFQEFTKQLEAEGMFEPSLPHVAYRLAEVIAMHVAGAA LIWHGYTFAGIAMLGVVQGRCGWLMHEGGHYSLTGNIAFDRAIQVACYGLGCGMSGAWWRNQHNKHHAT PQKLQHDVDLDTLPLVAFHERIAAKVKSPAMKAWLSMQAKLFAPVTTLLVALGWQLYLHPRHMLRTKHY DELAMLGIRYGLVGYLAANYGAGYVLACYLLYVQLGAMYIFCNFAVSHTHLPVVEPNEHATWVEYAANH TTNCSPSWWCDWWMSYLNYQIEHHLYPSMPQFRHPKIAPRVKQLFEKHGLHYDVRGYFEAMADTFANLD NVAHAPEKKMQ The mature Pavsa-5D sequence with mapped tryptic peptides (bold, underlined). For trypsin, all peptides selected were fully tryptic, i.e. contained no missed cleavages. As some of the peptides were adjacent in the sequence, these have been differentiated by single or double underline. RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V.
(217) The digestibility of His10::Pavsa-5D in SGF was assessed by LC-MRM-MS method as described above. Characterization and quantification of the targeted peptic peptides showed the rapid degradation of His10::Pavsa-5D. The pepsin digestion data has been presented in
(218) Eleven peptides were monitored by LC-MRM-MS spanning the length of the protein. A number of the peptides characterized and quantified after pepsin digestion were cleavage variants (
(219) TABLE-US-00033 TABLE 28 Percentage of each tryptic peptide remaining during pepsin time course Time (min) Peptide Sequence 5 10 15 30 60 DSYSYAAPPSAQLHEVDTPQEHDK 0.1 0.0 0.1 0.0 0.1 ELVIGDR 0.9 0.5 0.5 0.3 0.2 AYDVTNFVK 0.4 0.2 0.2 0.1 0.1 IIAYQVGTDATDAYK 0.5 0.3 0.3 0.2 0.2 SLPSRPVHK 21.6 6.6 8.1 6.4 3.7 ADLIADFQEFTK 19.4 6.9 7.6 6.0 2.7 QLEAEGMFEPSLPHVAYR 100.0 57.3 64.2 51.4 30.7 LQHDVDLDTLPLVAFHER 0.2 0.1 0.1 0.1 0.1 AWLSMQAK 31.6 9.1 12.9 10.8 5.7 HYDELAMLGIR 100.0 43.2 49.2 38.6 21.4 HGLHYDVR 43.3 29.2 32.6 28.4 18.8 GYFEAMADTFANLDNVAHAPEK 0.9 0.5 0.6 0.3 0.2 All peptides contained in SEQ ID NO: 5
(220) The rapid degradation of the His.sub.10::Pavsa-5D protein demonstrated by the rapid increase of peptic peptides was further demonstrated by rapid decline of tryptic peptides in trypsin digestion after pepsin digestion (combined pepsin-trypsin digestion). The majority (9/12) of the tryptic peptides monitored after the pepsin digest show a rapid decline in the first 5-10 min and then a further decline over the remainder of the 60 min duration experiment (
(221) Additionally, 99.9% of the full-length Pavsa-5D protein was digested within min and six out of 12 peptides remained less than 0.2% after 60 min of incubation in pepsin. The combined pepsin-trypsin assay showed a rapid decline in the tryptic peptides that were used as a proxy for the presence of intact protein. This LC-MRM-MS approach, using twelve peptides that spanned the intact protein, provided an understanding of the kinetics of digestion and the susceptibility of specific regions of the protein to proteolysis, and detected Pavsa-5D amounts as low as 7.80 femtomoles (injected on-column) which equates to 376 g on a protein scale.
Example 8. LC-MS/MS Analysis of Pyramimonas Cordata 6-Elongase Protein Stability
(222) This Example assess the in vitro stability of the Pyramimonas cordata 6-elongase (Pyrco-6E) protein in simulated gastric fluid (SGF) comprising the proteolytic enzyme, pepsin, and in combination with a novel pepsin-trypsin assay employing state-of-the-art mass spectrometric approaches to monitor the precise degradation products. The extent of protein digestion was evaluated by the appearance of peptic products and the disappearance of tryptic peptide products (as a proxy for intact protein). This Example shows that this integral membrane protein, when analyzed by LC-MS/MS, was readily digestible in pepsin and/or trypsin: >95% of the N-terminal and C-terminal regions of Pyrco-6E protein digested within 5 min and full-length protein was rapidly digested within 60 min of incubation in pepsin producing a suite of pepsin products <2,000 Da that spanned the entire peptide sequence.
(223) This 6-elongase gene was previously cloned from alga P. cordata. The Pyrco-6E protein was expressed as a His.sub.10::tag fusion in insect cell lines (Sf) infected with recombinant baculovirus constructed using pFastBac vector (Invitrogen, DE) and then purified. The vector contained coding sequences encoding a His-tag (His.sub.10) and a PreScission protease cleavage site (SLEVLFQGP) fused to the codon optimized Pyrco-6E gene to produce fusion protein His.sub.10::Pyrco-6E.
(224) The peptide fragments of His.sub.10::Pyrco-6E persisting after pepsin digestion for 120 min were characterized by untargeted LC-MS/MS, and shown in bold (wave underline shows the N-terminal His.sub.10::tag and protease cleavage site followed by methionine of native Pyrco-6E in the fusion protein):
(225) TABLE-US-00034 (SEQIDNO:18) MEFAQPLVAMAQEQYAAIDAVVAPAIFSA TDSIGWGLKPISSATKDLPLVESPTPLILSLLAYFAIVGSGLVYRKVFP RTVKGQDPFLLKALMLAHNVFLIGLSLYMCLKLVYEAYVNKYSFWGNAY NPAQTEMAKVIWIFYVSKIYEFMDTFIMLLKGNVNQVSFLHVYHHGSIS GIWWMITYAAPGGDAYFSAALNSWVHVCMYTYYFMAAVLPKDEKTKRKY LWWGRYLTQMQMFQFFMNLLQAVYLLYSSSPYPKFIAQLLVVYMVILLM LFGNFYYMKHHASK
(226) The peptide fragments present after trypsin digestion (for 16 h) were characterized by untargeted LC-MS/MS as shown below, in which bold indicates peptides identified with >9500 confidence, italics shows peptides identified with 50-95% confidence, and underlined indicates peptides identified with <50% confidence:
(227) TABLE-US-00035 (SEQIDNO:18) MEFAQPLVAMAQEQYAAIDAVVAPAIFSA TDSIGWGLKPISSATKDLPLVESPTPLILSLLAYFAIVGSGLVYRKVFP RTVKGQDPFLLKALMLAHNVFLIGLSLYMCLKLVYEAYVNKYSFWGNAY NPAQTEMAKVIWIFYVSKIYEFMDTFIMLLKGNVNQVSFLHVYHHGSIS GIWWMITYAAPGGDAYFSAALNSWVHVCMYTYYFMAAVLPKDEKTKRKY LWWGRYLTQMOMFQFFMNLLQAVYLLYSSSPYPKFIAQLLVVYMVTLLM LFGNFYYMKHHASK
(228) Pepsin-derived peptides that were identified with 95% c confidence and that yielded intense signals in the MS were selected for relative quantification. Twelve peptides were selected from the pepsin digestion of the His.sub.10::Pyrco-6E: protein, and four tryptic peptides, are summarized in Tables 29-30. The selected pepsin-derived peptides spanned the length of the protein.
(229) TABLE-US-00036 TABLE29 PeptideMRMtransitionsforPyrco-6Epepsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE FAQPLVA 5.16 373.220 2+ 444.220 b4+ 17.3 557.300 b5+ 656.370 b6+ MAQEQY 2.16 385.160 2+ 460.180 b4+ 17.9 588.240 b5+ 439.180 y3+ SATDSIGWGLKPISS 6.06 759.890 2+ 1057.600 y10+ 36.2 887.500 y8+ 944.520 y9+ WGLKPISS 4.92 444.250 2+ 531.310 y5+ 20.8 644.400 y6+ 701.420 y7+ VESPTPLIL 7.16 484.790 2+ 611.300 b6+ 22.8 724.390 b7+ 837.470 b8+ FAIVGSGL 6.48 382.220 2+ 431.270 b4+ 17.7 575.320 b6+ 632.340 b7+ LAHNVF 3.68 350.690 2+ 436.230 b4+ 16.2 535.300 b5+ 587.290 y5+ YVSKIYE 3.13 451.240 2+ 754.410 b6+ 21.1 639.330 y5+ 738.400 y6+ VSKIYE 2.30 369.700 2+ 591.350 b5+ 17.1 552.300 y4+ 639.330 y5+ YAAPGGDAY 3.30 442.690 2+ 632.260 b7+ 20.7 703.300 b8+ 579.240 y6+ YSSSPYPKF 4.43 538.260 2+ 651.350 y5+ 25.4 825.410 y7+ 912.450 y8+ YYMKHHASK 0.65 582.780 2+ 707.390 y6+ 27.6 838.440 y7+ 1001.500 y8+ Pyrco-6Esequence:.sup.b (SEQIDNO:4) MEFAQPLVAMAQEQYAAIDAVVAPAIFSATDSIGWGLKPISSATKDLPLVESPTPLILSLLAYFAIVGSGL VYRKVFPRTVKGQDPFLLKALMLAHNVFLIGLSLYMCLKLVYEAYVNKYSFWGNAYNPAQTEMAKVIWIFY VSKIYEFMDTFIMLLKGNVNQVSFLHVYHHGSISGIWWMITYAAPGGDAYFSAALNSWVHVCMYTYYFMAA VLPKDEKTKRKYLWWGRYLTQMQMFQFFMNLLQAVYLLYSSSPYPKFIAQLLVVYMVTLLMLFGNFYYMKH HASK .sup.bPyrco-6E sequence with mapped peptic peptides (bold, underlined). For pepsin, different cleavage variants were observed owing to the incomplete digestion and these peptides have been differentiated by single or double underline. RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V.
(230) TABLE-US-00037 TABLE30 PeptideMRMtransitionsforPyrco-6Etrypsinproducts Peptide RT Q1m/z z Q3m/z Fragment CE GQDPFLLK 5.12 459.258 2+ 732.429 y6+ 21.5 617.402 y5+ 260.190 y2+ LVYEAYVNK 4.14 549.780 2+ 886.430 y7+ 25.9 594.320 y5+ 723.360 y6+ YSFWGNAYNPAQTEMAK 6.07 989.440 2+ 1152.530 y10+ 47.5 1394.600 y13+ 875.430 y8+ YSFWGNAYNPAQTEM*AK 5.64 997.440 2+ 1168.530 y10+ 47.9 1410.630 y13+ 891.430 y8+ VIWIFYVSK 7.80 577.830 2+ 942.510 y7+ 27.3 756.430 y6+ 643.340 y5+ Pyrco-6Esequence:.sup.b (SEQIDNO:4) MEFAQPLVAMAQEQYAAIDAVVAPAIFSATDSIGWGLKPISSATKDLPLVESPTPLILSLLAYFAIV GSGLVYRKVFPRTVKGQDPFLLKALMLAHNVELIGLSLYMCLKLVYEAYVNKYSEWGNAYNPAQTEM AKVIWIFYVSKIYEFMDTFIMLLKGNVNQVSFLHVYHHGSISGIWWMITYAAPGGDAYFSAALNSWV HVCMYTYYFMAAVLPKDEKTKRKYLWWGRYLTQMQMFQFFMNLLQAVYLLYSSSPYPKFIAQLLVVY MVTLLMLFGNFYYMKHHASK RT, retention time (min); Q1 m/z, precursor ion mass-to-charge ratio (m/z); z, charge state; Q3 m/z, fragment ion m/z; CE, collision energy in V. The amino acid marked with * represents modified form: oxidation (Met).
(231) Digestibility of His.sub.10::Pyrco-6E in SGF was assessed by LC-MRM-MS method as described above. Characterization and quantification of the targeted peptic peptides showed the rapid degradation of His.sub.10::Pyrco-6E. The pepsin digestion data has been presented in
(232) The rapid degradation of the His.sub.10::Pyrco-6E protein demonstrated by the rapid liberation of peptic peptides was further confirmed by the decline of the single tryptic peptide after trypsin digestion (in the combined pepsin-trypsin digestion). Four of the peptides characterized and quantified after pepsin digestion were cleavage variants (
(233) TABLE-US-00038 TABLE 31 Percentage of each tryptic peptide remaining during pepsin time course Time (min) Peptide Sequence 5 10 15 30 60 GQDPFLLK 100.0 88.0 93.0 55.8 69.5 LVYEAYVNK 9.8 14.5 11.0 100.0 29.1 VIWIFYVSK 100.0 90.1 95.8 30.3 77.8 YSFWGNAYNPAQTEMAK 100.0 66.0 77.9 18.5 36.0 See SEQ ID NO: 4
(234) In the case of the His.sub.10::Pyrco-6E protein, fewer tryptic products were confidently identified and hence available for protein digestion monitoring. This was due to the decreased frequency and distribution of trypsin sites within the protein sequence resulting in few peptides amenable to LC-MS which were confined to the middle region of the protein. The first peptide monitored, GQDPFLLK (SEQ ID NO: 4), contained three potential pepsin cleavage sites (as indicated by the arrows) and it was expected that pepsin would cleave this peptide resulting in a decrease in peptide abundance over the time course of the pepsin digestion. After 5 min, however, the peptide peak area was noted to increase 4-fold, remain relatively constant over the next 10 min before proceeding to decline slowly over the next 45 min (
(235) Sequence Listing information:
(236) TABLE-US-00039 SEQIDNO:1,Picpa-3D MSKVTVSGSEILEGSTKTVRRSGNVASFKQQKTAIDTFGNVFKVPDYTI KDILDAIPKHCYERSLVKSMSYVVRDIVAISAIAYVGLTYIPLLPNEFL RFAAWSAYVESISCFGFGIWILGHECGHSAFSNYGWVNDTVGWVLHSLV MVPYFSWKFSHAKHHKATGHMTRDMVFVPYTAEEFKEKHQVTSLHDIAE ETPIYSVFALLFQQLGGLSLYLATNATGQPYPGVSKFFKSHYWPSSPVE DKKDYWYIVLSDIGILATLTSVYTAYKVFGFWPTFITWFCPWILVNHWL VFVTFLQHTDSSMPHYDAQEWTFAKGAAATIDREFGILGIIFHDIIETH VLHHYVSRIPFYHAREATECIKKVMGEHYRHTDENMWVSLWKTWRSCQF VENHDGVYMERNCNNVGVKPKDT SEQIDNO:2,Lackl-12D MSAVTVTGSDPKNRGSSSNTEQEVPKVAIDINGNVFSVPDFTIKDILGA IPHECYERRLATSLYYVERDIFCMLTTGYLTHKILYPLLISYTSNSIIK FTFWALYTYVQGLFGTGIWVLAHECGHQAFSDYGIVNDFVGWTLHSYLM VPYFSWKYSHGKHHKATGHMTRDMVFVPATKEEFKKSRNFFGNLAEYSE DSPLRTLYELLVQQLGGWIAYLEVNVTGQPYPDVPSWKWNHEWLTSPLF EQRDALYIFLSDLGILTQGIVLTLWYKKFGGWSLFINWFVPYIWVNHWL VFITFLQHTDPTMPHYNAEEWTFAKGAAATIDRKFGFIGPHIFHDIIET HVLHHYCSRIPFYNARPASEAIKKVMGKHYRSSDENMWKSLWKSFRSCQ YVDGDNGVLMERNINNCGVGAAEK SEQIDNO:3,Micpu-6D MCPPKTDGRSSPRSPLTRSKSSAEALDAKDASTAPVDLKTLEPHELAAT FETRWVRVEDVEYDVTNEKHPGGSVIFYMLANTGADATEAFKEFHMRSL KAWKMLRALPSRPAEIKRSESEDAPMLEDFARWRAELERDGFEKPSITH VAYRLLELLATFALGTALMYAGYPIIASVVYGAFFGARCGWVQHEGGHN SLTGSVYVDKRLQAMTCGFGLSTSGEMWNQMHNKHHATPQKVRHDMDLD TTPAVAFFNTAVEDNRPRGFSRAWARLQAWTFVPVTSGLLVQAFWIYVL HPRQVLRKKNYEEASWMLVSHVVRTAVIKLATGYSWPVAYWWFTEGNWI AYMYLFAHESTSHTHLPVVPSDKHLSWVNYAVDHTVDIDPSRGYVNWLM GYLNCQVIHHLEPDMPQFRQPEVSRREVPEAKKWGLNYKVLSYYGAWKA TESNLDKVGQHYYVNGKAEKAH SEQIDNO:4,Pyrco-6E MEFAQPLVAMAQEQYAAIDAVVAPAIFSATDSIGWGLKPISSATKDLPL VESPTPLILSLLAYFAIVGSGLVYRKVFPRTVKGQDPFLLKALMLAHNV FLIGLSLYMCLKLVYEAYVNKYSFWGNAYNPAQTEMAKVIWIFYVSKIY EFMDTFIMLLKGNVNQVSFLHVYHHGSISGIWWMITYAAPGGDAYFSAA LNSWVHVCMYTYYFMAAVLPKDEKTKRKYLWWGRYLTQMQMFQFFMNLL QAVYLLYSSSPYPKFIAQLLVVYMVTLLMLFGNFYYMKHHASK SEQIDNO:5,Pavsa-5D MPPRDSYSYAAPPSAQLHEVDTPQEHDKKELVIGDRAYDVTNFVKRHPG GKIIAYQVGTDATDAYKQFHVRSAKADKMLKSLPSRPVHKGYSPRRADL IADFQEFTKQLEAEGMFEPSLPHVAYRLAEVIAMHVAGAALIWHGYTEA GIAMLGVVQGRCGWLMHEGGHYSLTGNIAFDRAIQVACYGLGCGMSGAW WRNQHNKHHATPQKLQHDVDLDTLPLVAFHERIAAKVKSPAMKAWLSMQ AKLFAPVTTLLVALGWQLYLHPRHMLRTKHYDELAMLGIRYGLVGYLAA NYGAGYVLACYLLYVQLGAMYIFCNFAVSHTHLPVVEPNEHATWVEYAA NHTTNCSPSWWCDWWMSYLNYQIEHHLYPSMPQFRHPKIAPRVKQLFEK HGLHYDVRGYFEAMADTFANLDNVAHAPEKKMQ SEQIDNO:6,Pyrco-5E MASIAIPAALAGTLGYVTYNVANPDIPASEKVPAYFMQVEYWGPTIGTI GYLLFIYFGKRIMQNRSQPFGLKNAMLVYNFYQTFENSYCIYLFVTSHR AQGLKVWGNIPDMTANSWGISQVIWLHYNNKYVELLDTFEMVMRKKEDQ LSFLHIYHHTLLIWSWFVVMKLEPVGDCYFGSSVNTFVHVIMYSYYGLA ALGVNCFWKKYITQIQMLQFCICASHSIYTAYVQNTAFWLPYLQLWVMV NMFVLFANFYRKRYKSKGAKKQ SEQIDNO:7,Pavsa-4D MPPSAAKQMGASTGVHAGVTDSSAFTRKDVADRPDLTIVGDSVYDAKAF RSEHPGGAHFVSLFGGRDATEAFMEYHRRAWPKSRMSRFHVGSLASTEE PVAADEGYLQLCARIAKMVPSVSSGFAPASYWVKAGLILGSAIALEAYM LYAGKRLLPSIVLGWLFALIGLNIQHDANHGALSKSASVNLALGLCQDW IGGSMILWLQEHVVMHHLHTNDVDKDPDQKAHGALRLKPTDAWSPMHWL QHLYLLPGETMYAFKLLFLDISELVMWRWEGEPISKLAGYLFMPSLLLK LTFWARFVALPLYLAPSVHTAVCIAATVMTGSFYLAFFFFISHNFEGVA SVGPDGSITSMTRGASELKRQAETSSNVGGPLLATLNGGLNYQIEHHLF PRVHHGFYPRLAPLVKAELEARGIEYKHYPTIWSNLASTLRHMYALGRR PRSKAE SEQIDNO:8 TEPQTPQEWIDDLER SEQIDNO:9 EATECIK SEQIDNO:10 SCCAMQFVENHDGVYMER SEQIDNO:11 FHVGSLASTEEPVAADEGYLQLCAR SEQIDNO:12 SLEVLFQGP SEQIDNO:13 SVVAVIGLPNDPSVR SEQIDNO:14,His-taggedPicpa-3D HHHHHHHHSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEG YVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNENSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIG DGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK ENLYFQGGSSKVTVSGSEILEGSTKTVRRSGNVASFKQQKTAIDTFGNV FKVPDYTIKDILDAIPKHCYERSLVKSMSYVVRDIVAISAIAYVGLTYI PLLPNEFLRFAAWSAYVESISCFGFGIWILGHECGHSAFSNYGWVNDTV GWVLHSLVMVPYFSWKESHAKHHKATGHMTRDMVFVPYTAEEFKEKHQV TSLHDIAEETPIYSVFALLFQQLGGLSLYLATNATGQPYPGVSKFFKSH YWPSSPVEDKKDYWYIVLSDLGILATLTSVYTAYKVFGFWPTFITWFCP WILVNHWLVFVTFLQHTDSSMPHYDAQEWTFAKGAAATIDREFGILGII FHDIIETHVLHHYVSRIPFYHAREATECIKKVMGEHYRHTDENMWVSLW KTWRSCQFVENHDGVYMERNCNNVGVKPKDT SEQIDNO:15,His-taggedPyrco-5E MHHHHHHHHHHSLEVLFQGPMASIAIPAALAGTLGYVTYNVANPDIPAS EKVPAYFMQVEYWGPTIGTIGYLLFIYFGKRIMQNRSQPFGLKNAMLVY NFYQTFFNSYCIYLEVTSHRAQGLKVWGNIPDMTANSWGISQVIWLHYN NKYVELLDTFFMVMRKKEDQLSFLHIYHHTLLIWSWFVVMKLEPVGDCY FGSSVNTFVHVIMYSYYGLAALGVNCFWKKYITQIQMLQFCICASHSIY TAYVQNTAFWLPYLQLWVMVNMEVLFANFYRKRYKSKGAKKQ SEQIDNO:16,His-taggedPavsa-4D MHHHHHHHHHHSLEVLFQGPMPPSAAKQMGASTGVHAGVIDSSAFTRKD VADRPDLTIVGDSVYDAKAFRSEHPGGAHFVSLFGGRDATEAFMEYHRR AWPKSRMSRFHVGSLASTEEPVAADEGYLQLCARIAKMVPSVSSGFAPA SYWVKAGLILGSAIALEAYMLYAGKRLLPSIVLGWLFALIGLNIQHDAN HGALSKSASVNLALGLCQDWIGGSMILWLQEHVVMHHLHTNDVDKDPDQ KAHGALRLKPTDAWSPMHWLQHLYLLPGETMYAFKLLFLDISELVMWRW EGEPISKLAGYLFMPSLLLKLTFWARFVALPLYLAPSVHTAVCIAATVM TGSFYLAFFFFISHNFEGVASVGPDGSITSMTRGASFLKRQAETSSNVG GPLLATLNGGLNYQIEHHLFPRVHHGFYPRLAPLVKAELEARGIEYKHY PTIWSNLASTLRHMYALGRRPRSKAE SEQIDNO:17,His-taggedPavsa-5D MHHHHHHHHHHSLEVLFQGPMPPRDSYSYAAPPSAQLHEVDTPQEHDKK ELVIGDRAYDVTNFVKRHPGGKIIAYQVGTDATDAYKQFHVRSAKADKM LKSLPSRPVHKGYSPRRADLIADEQEFTKQLEAEGMFEPSLPHVAYRIA EVIAMHVAGAALIWHGYTFAGIAMLGVVQGRCGWLMHEGGHYSLTGNIA FDRAIQVACYGLGCGMSGAWWRNQHNKHHATPQKLQHDVDLDTLPLVAF HERIAAKVKSPAMKAWLSMQAKLFAPVTTLLVALGWQLYLHPRHMLRTK HYDELAMLGIRYGLVGYLAANYGAGYVLACYLLYVQLGAMYIFCNFAVS HTHLPVVEPNEHATWVEYAANHTTNCSPSWWCDWWMSYLNYQIEHHLYP SMPQFRHPKIAPRVKQLFEKHGLHYDVRGYFEAMADTFANLDNVAHAPE KKMQ SEQIDNO:18,His-taggedPyrco-6E MHHHHHHHHHHSLEVLFQGPMEFAQPLVAMAQEQYAAIDAVVAPAIFSA TDSIGWGLKPISSATKDLPLVESPTPLILSLLAYFAIVGSGLVYRKVFP RTVKGQDPFLLKALMLAHNVFLIGLSLYMCLKLVYEAYVNKYSFWGNAY NPAQTEMAKVIWIFYVSKIYEFMDTFIMLLKGNVNQVSFLHVYHHGSIS GIWWMITYAAPGGDAYFSAALNSWVHVCMYTYYFMAAVLPKDEKTKRKY LWWGRYLTQMQMFQFFMNLLQAVYLLYSSSPYPKFIAQLLVVYMVILLM LFGNFYYMKHHASK