METHOD AND DEVICE FOR PROTEIN PREPARATION

Abstract

The present invention provides a method of preparing a sample comprising one or more proteins of interest, the method comprising: providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium; exposing said sample to a mild precipitant to cause precipitation of said proteins; during or after the precipitation step, bringing said sample into contact with a matrix adapted to capture said precipitated proteins and prevent excessive aggregation of precipitated protein particles; and washing the matrix with captured precipitated proteins to remove the surfactant. A sample preparation device to carry out the same is also provided.

Claims

1-48. (canceled)

49. A method of preparing a sample comprising one or more proteins of interest, the method comprising the ordered steps of: providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium; exposing said sample to a mild precipitant to cause precipitation of said proteins; wherein the mild precipitant causes the protein solubilised with surfactant to precipitate to form a suspension of particles of protein, wherein said particles of protein are sensitive to protease digestion; during or after the precipitation step, bringing said sample into contact with a matrix that is a depth filter adapted to capture said precipitated protein particles and prevent excessive aggregation of precipitated protein particles to thereby allow a protease to digest the protein particles in situ; washing the matrix containing the captured precipitated protein particles to remove the surfactant; and after washing the matrix, digesting the protein particles in situ.

50. The method of claim 49 wherein the matrix is porous or fibrous material which is able to be penetrated by the medium comprising the proteins and which permits the precipitated proteins to be reversibly captured by the matrix.

51. The method of claim 49 wherein the matrix comprises one or more of quartz, glass, polymer and cellulose materials.

52. The method of claim 49, wherein the matrix: is adapted to capture and retain protein particles of 10 μm or smaller; is substantially inert with respect to the proteins in the sample such that undesirable reactions between the matrix and the sample are avoided; is able to reversibly capture proteins from the sample; and does not bind to, and therefore retain, the surfactant.

53. The method of claim 49 wherein the mild precipitant is an acid.

54. The method of claim 53 wherein the mild precipitant is phosphoric acid.

55. The method of claim 49 wherein the matrix is permeated with an aqueous methanolic solution, prior to introduction of the precipitated protein.

56. The method of claim 49, further comprising exposing the matrix with protein particles captured thereon to a protease to generate fragments of the proteins.

57. The method of claim 56, further comprising the step of eluting the proteins and/or fragments thereof from the matrix.

58. The method of claim 57, wherein proteins or protein fragments are eluted from the matrix and then pass to a secondary matrix.

59. The method of claim 58, comprising eluting the protein fragments from the secondary matrix using a suitable elution solution.

60. The method of claim 55, wherein the protein solubilised with surfactant immediately forms a suspension of particles of protein after the addition of the solution comprising the mild precipitant and solubilised protein to the aqueous methanolic solution permeating the matrix.

61. The method of claim 49, wherein the method does not comprise the use of urea.

62. A method of preparing a sample comprising one or more proteins of interest, the method comprising the ordered steps of: providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium; exposing said sample to a precipitant, wherein said precipitant solubilises the surfactant, causes precipitation of said proteins as a suspension of particles of protein, and does not render the precipitated proteins insensitive to protease digestion; during or after the precipitation step, bringing said sample into contact with a matrix that is a depth filter having a network of pore channels adapted to capture said precipitated protein particles throughout the depth filter and thereby prevent excessive aggregation of precipitated protein particles at a surface of initial contact between the precipitated protein particles and the depth filter, and wherein further said matrix does not bind the solubilised surfactant; and digesting the captured precipitated protein particles in situ using a protease.

63. The method of claim 62, wherein the matrix is porous or fibrous material which is able to be penetrated by the medium comprising the proteins and which permits the precipitated proteins to be reversibly captured by the matrix.

64. The method of claim 62, wherein the matrix comprises one or more of quartz, glass, polymer and cellulose materials.

65. The method of claim 62, wherein the matrix: is adapted to capture and retain protein particles of 10 μm or smaller; is substantially inert with respect to the proteins in the sample such that undesirable reactions between the matrix and the sample are avoided; and is able to reversibly capture proteins from the sample.

66. The method of claim 62, wherein the precipitant is an acid.

67. The method of claim 66, wherein the acid is phosphoric acid.

68. The method of claim 62, wherein the matrix is permeated with an aqueous methanolic solution prior to introduction of the precipitated protein.

69. The method of claim 62, further comprising the step of eluting fragments of the digested protein particles from the matrix.

70. The method of claim 69, wherein the fragments of the digested protein particles are eluted from the matrix and then pass to a secondary matrix.

71. The method of claim 70, comprising eluting the protein fragments from the matrix using a suitable elution solution.

72. The method of claim 68, wherein the protein solubilised with surfactant immediately forms a suspension of particles of protein after the addition of the solution comprising the precipitant and solubilised protein to the aqueous methanolic solution permeating the matrix.

73. The method of claim 62, further comprising the step of washing the matrix with the captured precipitated protein particles to remove the surfactant, wherein the washing step is performed after the precipitation step and before the step of digesting the protein particles in situ.

74. The method of claim 62, further comprising the step of washing the matrix with the captured precipitated protein particles to remove the surfactant, wherein the washing step is performed after the precipitation step and before the step of digesting the protein particles in situ, and wherein an aqueous methanolic solution is used in the washing step.

75. The method of claim 62, wherein the precipitated protein particles are captured throughout the depth filter on the basis of particle size, and wherein the step of digesting the captured protein particles in situ comprises digesting the protein particles to fragments having sizes which are smaller than the network of pore channels in the depth filter matrix to facilitate elution of the digested protein particle fragments from the depth filter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1. Suspension Trapping (STrap)-based processing of Hela SOS lysate. (a) The proteins are solubilised in the presence of high concentration of SOS, reduced and alkylated. The sample is acidified and introduced into the STrap tip (S-tip). The detergent and other contaminants are removed in the flow-through. The protein suspension is trapped in the S-tip and, after the introduction of trypsin, the digestion is performed for 30 min at 47° C. The key steps in the STrap processing are visualized with the use of the Coomasie-stained polyacrylamide gel. (b) The STrap method allows rapid reactor-type processing of the SDS-solubilised protein material. An average number of identified proteins from triplicate 240-min LC-MS/MS runs is shown. (c) Comparison of our HeLa proteome dataset obtained using RP-RP STrap processing with complete HeLa proteome by Nagaraj at al..sup.11. The percentage of proteins with the appropriate Gene Ontology annotations is shown.

[0130] FIG. 2. Using STrap for immunoprecipitation (IP) profiling with low amounts of antibodies. (a) The IP was performed using 2 μg of anti-cyclin L 1 (CCNL 1) antibody, the eluate was processed with the STrap methodology. The key steps of the STrap processing are visualized with the use of the silver-stained polyacrylamide gel. (b) The eluted proteins from anti-CCNL1 and control antibody pull-down experiments were processed with the STrap methodology, identified and quantified by LC-MS/MS. The log.sub.10 values of the identified protein intensities are mapped onto the relevant axes.

[0131] FIG. 3. STrap-tip processing unit. (a) The parts of the STrap-tip Spin-unit. (b) Assembled Spin-unit. (c) The STrap-tip with quartz/C.sub.18 plugs stack.

[0132] FIG. 4. Depiction of the STrap-tip-based sample processing. To the STrap-tip (1) the Strapping buffer is added (2), the acidified sample is added to the Strapping buffer (3) and the protein suspension is formed (3A). After the centrifugation, the protein suspension is trapped in the quartz stack (38). Following the washes with the Strapping buffer (4) and water (5) an enzyme is added and the STrap tip is closed with a filter tip (6). After the incubation with an enzyme, the STrap tip is washed with the Ammonium Bicarbonate (AmBic) and TFA solutions (7, 8). The peptides, captured by the reversed phase plugs, are eluted with the Elution solution (9).

[0133] FIG. 5. STrap-tip processing of the sub-μg protein loads from HeLa SDS lysate. 120-min triplicate LC-MS/MS runs were used for protein identification.

[0134] FIG. 6. STrap-tip processing of the enriched membrane protein fraction from HeLa cells. (a) The key steps in the STrap processing are visualized with the use of the Coomasie-stained polyacrylamide gel. (b) Comparison of the obtained enriched membrane protein dataset with the complete HeLa proteome by Nagaraj at al..sup.11 The percentage of proteins with the relevant Gene Ontology annotations is shown.

[0135] FIG. 7. STrap-tip processing of the HeLa lysate using Lys-C protease (Wako). (a) The use of Lys-C protease identifies comparable to the tryptic processing protein numbers (based on triplicate 240-min LC-MS/MS runs). (b) Venn diagram shows the overlap between the proteins identified with trypsin and Lys-C proteases.

[0136] FIG. 8. An example of the STrap processing device with a larger loading capacity—the STrap-tube unit. (a) The parts of the STrap-tube Spin-unit. (b) Assembled Spin-unit. (c) The STrap-tube with quartz/C.sub.18 disk assembly. Five quartz and three C.sub.18 disks are cut out of the corresponding membranes using 4.5 mm ID tubing. The disks are inserted/pressed down with a custom-made pusher into the 0.5 ml sample tube which was punctured with a 25G needle in the bottom and lid parts.

[0137] FIG. 9. Estimation of the particle size range formed during the STrap processing. Hela SDS lysate was acidified and precipitated in the neutral methanolic solution and passed through the filter paper stacked in a pipette tip. The trapped protein material was eluted with 2×sample loading buffer and visualized with the use of the Coomasie-stained polyacrylamide gel. EEs

[0138] FIG. 10. Comparison of borosilicate glass (GF/D) filter with quartz (MK360) filter performance for the STrap processing of cellular lysates. The key steps are visualized with the use of the Coomasie-stained polyacrylamide gel.

[0139] FIG. 11. Cys-STrap proteomics analysis of the MGH-U3 cell lysate. The Venn diagram shows the peptide numbers identified by mass spectrometry in each of the distinct digest fractions.

[0140] FIG. 12. Serum preparation for the downstream processing with the STrap methodology. We observe removal of the major portion of serum albumin in the precipitated fraction. The supernatant is taken for further processing with the Cys-STrap method.

DETAILED DESCRIPTION

[0141] A typical bottom-up proteomic experiment is based on tryptic proteolysis with subsequent characterization of the generated peptide products by mass spectrometry (MS).sup.1. In order to obtain a thorough map of proteins, detergents (surfactants), amphipathic by nature, which facilitate solubilisation of hydrophobic proteins, are deployed. The conventional detergents are not compatible with mass spectrometry and must be eliminated from the samples prior to MS analysis. While attempts have been made to introduce mass spectrometry-friendly surfactants.sup.2, 3, such surfactants have not become widely accepted allegedly due to their high cost and suboptimal performance. Brought into the biochemistry spotlight in 1965.sup.4, sodium dodecyl sulphate (SDS), an anionic surfactant, is the most widely used detergent for protein separation and solubilisation. The most common analytical utility of SDS is in separation of proteins by means of polyacrylamide gel electrophoresis (SDS-PAGE).sup.5. The protein-containing gel bands may be cut out and in-gel digestion performed6. However, the overall containing gel bands may be cut out and in-gel digestion performed.sup.6. However, the overall value of such a procedure for proteome profiling and quantitation is limited due to such adverse effects as partial protein digestion, artifactual modifications, poor peptide recovery and the difficulties in processing of large numbers of gel bands if large-scale comparative profiling across multiple samples is needed. During recent years, the Filter Aided Sample Preparation Method (FASP) has gained popularity as one of the key tools for gel-free proteomic processing of the cellular and tissue SOS-extracted material7. The method, performed in ultrafiltration devices, is based on repetitive steps involving disruption of protein-SOS micelles by the chaotropic action of urea, removal of SOS and urea, and on-filter enzymatic cleavage of proteins which should result in adequate peptide yield and purity. The important analytical property of such a procedure is its unbiased nature, i.e. its ability to supply objective coverage of the hydrophobic (e.g. associated with membranes) and hydrophilic (e.g. cytoplasmic) cellular protein content. The FASP method, however, is very time-consuming, dependent on batch-to-batch reliable performance of the commercial spin-filter units, requires careful implementation and control to prevent drying-out or damage of the spin-filter membrane, and is disconnected from the clean-up step. The aim of the present invention work was to create a simple, efficient and reproducible ‘workhorse’ sample preparation tool, compatible with small sample amounts, which would combine the proven power of the SDS-based protein extraction with rapid detergent removal, protein digestion and in-situ clean-up of the peptides.

[0142] The key concept underpinning the basic STrap method design which we describe here is an instant creation of the fine protein particulate suspension, still susceptible to a protease action, from an SDS-solubilised protein solution, along with limiting the extent of the further aggregation of the suspension by its entrapment in the stack of an in-depth filtration material. This is achieved by adding the acidified protein-SDS mixture to a methanolic solution at a near-neutral pH in an S-tip incorporating depth filter and reversed phase membrane compartments (FIG. 3). The addition results in immediate creation of the fine protein suspension which is then captured by the depth filtration plugs. The SDS monomers which are soluble in the methanolic solution and other contaminants are washed away. The entrapped material is subsequently digested by an introduced protease. When the digestion is complete, the peptide products are transferred into and captured by the hydrophobic C.sub.18 bottom part of the S-tip plug assembly, desalted, eluted and concentrated ready for the following liquid chromatography—tandem mass spectrometry (LC-MS/MS) run (FIG. 4). An example of the ST rap tryptic processing of 30 μg protein from HeLa lysate is presented in FIG. 1a. Complete capture of the loaded protein material in the S-tip can be inferred from its evident absence in the flow-through fraction. After the 30-min incubation with trypsin at 47° C., the digest products are cleaned-up and eluted. No noticeable protein is left in the S-tip after the peptide elution step. In this case, approximately 3500 HeLa proteins are consistently identified in a 4-hour LC-MS/MS run injecting one third of the produced peptide amount. As the digestion happens in a limited volume and at elevated temperature, the tryptic enzymatic reaction is accelerated.sup.8,9, with proteolysis being partially completed in only a few minutes allowing identification of more than two thousand proteins even after such a short time (FIG. 1 b). By replacing the C.sub.18 with C.sub.8 plugs and in-tip reversed phase fractionation of the peptides into four fractions which were consequently analysed by LC-MS/MS, mimicking the two-dimensional reversed phase-reversed phase (RP-RP) chromatographic separation approach.sup.11, we identified almost 5000 HeLa proteins in about 11 hours of total acquisition time. The overall representation of the characteristic protein groups (membrane, cytoplasmic and nuclear) in our data as indicated by Gene Ontology (GO) analysis was similar to that provided in the most complete exhaustive HeLa proteome data set of about 10,000 proteins obtained by extensive protein fractionation, FASP processing, and a total acquisition time of 288 hours.sup.11 (FIG. 1c) confirming the unbiased output of our technique.

[0143] To determine whether there are any unreasonable sample losses while working with sub-μg protein loads we processed amounts spanning the two orders of magnitude—75 ng, 750 ng and 7.5 μg of HeLa lysate. No disproportionate decrease in protein identifications was observed (FIG. 5), with 763, 1643 and 2096 average number of proteins identified, respectively, using 120-min triplicate LC-MS/MS runs, similar to the results reported for the FASP methodology.sup.12.

[0144] In order to show the applicability of our method towards the analysis of less complex low protein amount samples, we performed an immunoprecipitation experiment using 2 of anti-cyclin L1 (CCNL1) polyclonal antibody which is directed against the N-terminal part of this transcriptional regulator thus leaving the C-terminal RS domain of CCNL1 accessible to interactions. The bound macromolecular complexes were eluted with 5% SDS and processed using the STrap methodology (FIG. 2a). As the result, in the pool of about 1000 identified proteins-background molecules and potential specific interactors-we could easily pinpoint, besides the targeted CCNL1, cyclin-dependent kinase 11B (CDK11B), the key CCNL1 target.sup.13 (FIG. 2b), with 38% and 39% of the protein sequences covered, respectively. The experiment demonstrated that utilizing low amounts of antibodies and the STrap methodology could be efficiently used to uncover protein-protein interactions. One of the most important characteristics of any bottom-up proteomics sample preparation method is its ability to process membrane proteins which due to their inherent hydrophobicity are not easily enzymatically cleaved in aqueous environments. We used a modified procedure of Bordier.sup.14 for preparation of an enriched fraction of membrane proteins from HeLa cells. The procedure is based on the ability of a solution of the non-ionic detergent Triton X-114 to extract protein material at cold temperatures with subsequent separation at temperatures exceeding 20° C. into an aqueous phase, containing hydrophilic proteins, and detergent phase, containing membrane proteins. The obtained enriched fraction of membrane proteins together with Triton X-114 detergent was then solubilised with 15% basic SDS solution—this solubilisation step made it possible to flawlessly process the sample by the STrap protocol using tryptic digestion for one hour at 47° C. (FIG. 6a). As the result, more than 3000 proteins were identified with 45% of the proteins being categorized by GO annotation as being part of a membrane as compared with only 25% in the whole HeLa proteome (FIG. 6b). The experiment also demonstrates the STrap capability to process samples with high SOS content.

[0145] Even though our method was originally optimized using trypsin as the most common robust protease with high primary specificity and thermal stability, we reasoned that deployment of the STrap concept could also be helpful while working with additional enzymes. However, the other enzymes such as Lys-C, for example, perform best at the manufacturer's recommended temperature conditions. Nonetheless, besides more lengthy incubation period with an enzyme, the core of the STrap procedure remains unaltered and the advantage of the overall improvement in sample preparation times is retained. To demonstrate the idea's applicability, we performed the STrap procedure using 30 μg of HeLa lysate and Lys-C endoproteinase with 4-hour incubation at 37° C. in a humidified chamber. The Lys-C performance in terms of a number of protein identifications was similar to that of trypsin—more than 3500 proteins were routinely identified using 240-min LC-MS/MS runs injecting one third of the resultant peptides amount (FIG. 7a) and about 4000 proteins were identified using the data from three replicate runs (FIG. 7b).

[0146] In the described ST rap-tip format, our method provides the means for rapid processing of the SOS-solubilised protein material from about 50 μg down to sub-microgram amounts. Taking into account the fact that the constantly evolving modern LC-MS/MS systems with high sensitivity, accuracy and sequencing speed require only several micrograms of peptides for comprehensive proteomics profiling.sup.15,16 the tip implementation of the STrap method is going to be sufficient for many a routine proteomics task. If necessary, however, larger protein quantities may be processed by adhering to the explained STrap principles and designing appropriate in-depth trapping/processing units with an increased surface area. An example of the larger capacity unit—STrap-tube—is presented in FIG. 8. This work outlines the concept and demonstrates the practical applicability of the Suspension Trapping (STrap) methodology facilitating proteomics analysis of the various SDS-solubilised protein mixtures—cellular lysates, membrane preparations and immunoprecipitates. Importantly, our method provides rapid, unproblematic, reproducible and simple sample processing capability for the low microgram protein quantities—the ‘precarious’ area working in which formerly required, in addition to technical dexterity, considerable inputs of time and endeavour.

[0147] Methods

[0148] The S-Tip Design

[0149] The basic trapping and clean-up S-tip device is made either from the quartz fibre (MK360, Munktell or QM-A, Whatman) filters, borosilicate glass fibre (GF/D, Whatman) filter or their combination and reversed phase membrane (Empore C.sub.18, 3M) disk plugs stacked together in a pipette tip (D200, Gilson) using gauge 14 blunt end needle (Z261394, Sigma). Eleven MK360 quartz plugs or nine borosilicate GF/D glass plugs or combination of either six borosilicate GF/D glass and five quartz MK360 plugs or six QM-A quartz and five MK360 quartz plugs, and three or four C.sub.18 plugs are forced into the 200 μl pipette tip end with the aid of a pusher—the piece of 1/16″ OD PEEK tubing (1535, Upchurch Scientific). in order to compact the plugs and make them adhere to each other, the stack is further pressed down and compressed with a piece of the PEEK tubing several times” Originally, the quartz fibre filter from Munktell (MK 360) was chosen as a preferred in-depth filtration material because of its pure quartz composition, ability to trap particles down to sub-micron range, absence of binders and heat pre-treatment which provide an adequate near-contaminant-free trapping matrix environment. However, the borosilicate glass binderless fibre filter from Whatman (GF/D) also proved to work well for the described applications. FIG. 10 shows a comparison of the results of using MK360 and GF/D filters in the standard STrap protocol as visualised by Coomasie-stained poiyacrylamide gel electrophoresis. Even though the borosilicate glass fibre displays a somewhat stronger peptide binding in comparison to the quartz material, the use of GF/D filter with larger pore sizes and loading capacity may be beneficial wilen working with the protein loads exceeding 30 μg. Alternatively, the quartz QM-A (Whatman) filter with larger than MK360 material pore sizes could be utilized. The underlying Empore reversed phase material gives mechanical support for the upper plugs, captures stray particles as well as a shed fibre material, and, in addition, serves as a medium for the final clean-up of the peptides.

[0150] Cell Solubilisation and Lysate Processing

[0151] A Hela cell pellet (8 million cells) was lysed in excess of a lysis solution (5% SDS in 50 mM TRIS-HCl, pH 7.6) at room temperature (RT). To shear the DNA, the sample was sonicated briefly with a probe sonicator. Then, dithiothreitol (DTT) (stock solution of 1M in H.sub.2O) was added to a final concentration of 20 mM. The extract was heated at 95° C. for 5 min and then clarified by centrifugation at 12,100×g for 10 min. Protein concentration was measured by tryptophan fluorescence as described previously.sup.15. Iodoacetamide (IAA) (stock solution of 0.9 M in H.sub.2O) was added to a final concentration of 150 mM. Following incubation for 15 min in the dark, the lysate was ready for further processing by the STrap method.

[0152] Basic Sample Processing Procedure by Strap Method

[0153] The required protein amounts (75 ng-30 μg), in triplicates, were prepared by appropriate dilutions of the alkylated SDS Hela lysate with the lysis solution to a final volume of 18 μL and processed adhering to the STrap protocol (see Exemplary Suspension Trapping Methodology) using 30-min tryptic digestion at 47° C. performed in the 0-tubes pre-heated to 47° C.

[0154] Mass Spectrometry and Data Analysis

[0155] Peptides were separated online by reversed-phase capillary liquid chromatography (LC) using RSLCnano system (Dionex) connected to a 40-cm capillary emitter column made in-house (inner diameter 75 μm, packed with 3 μm Aqua C.sub.18 media). The chromatography system was hyphenated with a linear quadrupole ion trap-orbitrap (LTQ-Orbitrap) Velos mass spectrometer (Thermo). The total acquisition times used for basic STrap processing were either 120, 200 or 240 min, the major part of the chromatographic gradient was 2%-32% acetonitrile (ACN) in 0.2% formic acid. Survey MS scans (scan range of 300-1500 amu) were acquired in the orbitrap with the resolution set to 60 000. Up to 20 most intense ions per scan were fragmented and analysed in the linear trap. Data were processed against a Uniprot human protein sequence database (October, 2012) with MaxQuant 1.3.0.5 software.sup.17 and Andromeda search engine.sup.18. The mass tolerance for MS scan was set to 7 ppm, the fragment mass tolerance for MS/MS was set to 0.5 Th. Carbamidomethylation of cysteine was set as a fixed modification, with protein N-terminal acetylation and oxidation of methionine as variable modifications, two missed cleavages, and at least 1 unique peptide for valid protein identification. The maximum protein and peptide false discovery rates were set to 0.01. Bioinformatics analysis of Gene Ontology (GO) features was undertaken with Perseus 1.3.0.4 (www.maxquant.org).

[0156] Preparation of the Enriched Membrane Protein Fraction with Triton X-114

[0157] To a Hela cell pellet containing 8 million cells, 500 μL of 1% Triton X-114 ice-cold solution in phosphate-buffered saline (PBS) pH 7.4 was added. The sample was rotated in the cold room for 10 min and centrifuged at 5000×g for 10 min at 5° C. The supernatant, containing mostly membrane and cytoplasmic proteins, was removed, placed at 45° C. for 10 min, and then centrifuged at 12,100×g for 10 min at RT. The upper fraction was removed. The protein pellet and the Triton X-114 lower fraction were washed once by reconstitution in 300 μL of PBS preheated to 45° C. and centrifugation at 12,100×g for 7 min at RT. The upper fraction was removed and the lower fraction and the pellet were solubilised with 200 μL of 15% SDS in 50 mM TRIS-HCl, pH 7.6. DTT was added to a final concentration of 20 mM and the sample was heated at 95° C. for 5 min. The solution was clarified by centrifugation at 12,100×g for 10 min (as Triton X-114 interferes with tryptophan fluorescence measurement, for estimation of protein concentration by this method proteins in a sample aliquot were precipitated and recovered into 5% SDS 50 mM TRIS-HCl, pH 7.6 buffer). 25-μg protein aliquots were taken and IAA added to a final concentration of 150 mM. Following incubation for 15 min in the dark, the samples were ready for further processing by the quartz-based STrap method using a tryptic incubation for one hour at 47° C. The peptides were analysed by the 240-min LC-MS/MS method.

[0158] Immunoprecipitation

[0159] Immunoprecipitation using polyclonal rabbit anti-cyclin L1 (anti-CCNL1) antibody (A302-058A, Bethyl Laboratories) and control 1 gG from rabbit serum (15006, Sigma) was performed as outlined below. HeLa cell pellet (25 million cells) was extracted with 1.0 ml of Radio-Immunoprecipitation Assay (RIPA) buffer containing protease inhibitors (Complete™ Mini Protease Inhibitor Cocktail Tablet, EOTA-free, Roche). The extract was cleared by centrifugation at 12,100×g for 10 min. A 2 μg aliquot of antibody was added to the extract. The mixture was rotated at RT for 30 min. Afterwards, 30 μl of Protein G magnetic beads (Oynabeads, Life Technologies) was added and the mixture was rotated for another 30 min. After removal of the extract and a wash with RIPA buffer, bound proteins were eluted by incubating the beads with 30 μl of 5% SDS 50 mM TRIS-HCl, pH 7.6 buffer containing 20 mM DTT at 90° C. for 5 min. Alkylation was performed by adding IAA to the final concentration of 150 mM. After processing by the quartz-based ST rap method, the resultant peptides were analysed by LC-MS/MS using the 200-min acquisition method.

[0160] Reversed Phase Sample Fractionation with STrap

[0161] The modified quartz-based STrap tip was created by substituting the C.sub.18 with C.sub.8 reversed phase material. 50 μg of HeLa lysate was introduced into the STrap tip, digested with trypsin at 47° C. for 30 min using the 0-tubes pre-heated to 47° C. and the peptides were transferred to the reversed phase plug compartment according to the basic STrap protocol. Afterwards, four peptide fractions were obtained by consecutive elutions with 50 μl of 5% ACN in water, 10% ACN in water, 15% ACN in water and 60% acetonitrile in 0.5% formic acid (FA). The concentrated peptide fractions were chromatographically separated online with the following 140 min gradients-2-15% ACN in 0.2% FA, 2-21% ACN in 0.2% FA, 9-25% ACN in 0.2% FA, 12-36% ACN in 0.2% FA, respectively, and analysed by MS/MS similarly to the described above.

[0162] Lysyl Endopeptidase (Lys-C) and STrap

[0163] The samples, 30 μg of Hela lysate, were processed similarly to the quartz-based STrap protocol except that, instead of trypsin, Lys-C protease (125-05061, Wako) was used (0.033 μg/μl in Tris-HCl pH 9.0 buffer) and the incubation was performed for 4 hours at 37° C. in a humidified chamber. The resultant peptides were analysed by LC-MS/MS using the 240-min acquisition method as described above.

[0164] Exemplary Suspension Trapping Methodology (STrap)

[0165] Tryptic Digest, Maximum Load of 50 μg of Total Protein

[0166] 1. Materials

[0167] Solutions and Reagents [0168] Milli-Q water (H.sub.2O) [0169] AmBic (Ammonium Bicarbonate) solution: 50 mM NH.sub.4HCO.sub.3 in H.sub.2O [0170] Lysis buffer: 5% (w/v) sodium dodecyl sulfate (SDS), 50 mM Tris/HCl pH 7.6 [0171] STrapping buffer: 90% methanol, 100 mM Tris/HCl pH 7.1 [0172] Phosphoric acid stock solution: 12.15% in H.sub.2O [0173] OTT stock solution: 1 M dithiothreitol in H.sub.2O, prepared on the day of experiment [0174] IAA stock solution: 0.9 M iodoacetamide in H.sub.2O, prepared on the day of experiment [0175] Trypsin solution: 0.033 μg/μl of trypsin (03708985001, Roche or V5111, Promega) in 50 mM NH.sub.4HCO.sub.3, prepared prior to starting the STrap processing step and kept on ice [0176] TFA solution 1: 0.5% trifluoroacetic acid in H.sub.2O [0177] TFA solution 2: 10% trifluoroacetic acid in H.sub.2O [0178] Elution solution: 70% acetonitrile, 0.5% formic acid in H.sub.2O [0179] FA solution: 0.2% formic acid in H.sub.2O

[0180] Equipment [0181] Bench-top centrifuge (for example MiniSpin, Eppendorf) [0182] Probe Sonicator (for example Soniprep 150, MSE) [0183] Heating block suitable for handling 1.5 ml microtubes (for example PHMT, Grant Bio) [0184] Plastic Syringe, 20 ml (for example 301031, BO) with a custom adapter to fit into 200 μl pipette tips [0185] Vacuum Concentrator (for example SpeedVac, Thermo)

[0186] STrap-tip (S-tip): S-tip is made either from the quartz fibre (MK360, Munktell or QM-A, Whatman) filters, borosilicate glass fibre (GF/D, Whatman) filter or their combination and reversed phase membrane (Empore C.sub.18, 3M) disk plugs stacked together in a pipette tip (D200, Gilson) using gauge 14 blunt end needle (Z261394, Sigma). Eleven MK360 quartz plugs or nine borosilicate GF/D glass plugs or combination of either six borosilicate GF/D glass and five quartz MK360 plugs or six QM-A quartz and five MK360 quartz plugs, and three or four C.sub.18 plugs are forced into the 200 μl pipette tip end with the aid of a pusher—the piece of 1/16″ OD PEEK tubing (1535, Upchurch Scientific). In order to make the plugs adhere to each other, the stack is further pressed down and compacted with a piece of the 1/16″ OD PEEK tubing several times.

[0187] 0-tube: A 1.5 ml microcentrifuge tube (72.690.001, Sarstedt) with an opening punctured in the tube's lid (alternatively, a pipette tip lid-adapter for microcentrifuge tubes could be used). The S-tip and O-tube comprise the Spin-unit.

[0188] Filter tips, 10 μl (TF-300-R-S, Axygen).

[0189] 2. Methods

[0190] 2.1 Cell Lysis and Reduction of Cysteine Residues

[0191] Cells are lysed in excess of the Lysis buffer (ca. 1:8-1:10 sample-to-Lysis buffer volume ratios) at room temperature. To shear the DNA, the lysate is shortly sonicated using a probe sonicator. DTT stock solution is added to a final concentration of 20 mM. The extract is heated up at 95° C. for 5 min. The extract is clarified by centrifugation at 12,100×g for 10 min. Protein concentration could be measured by tryptophan fluorescence (Wisniewski, J. R., Dus, K. & Mann, M. Proteomics Clin Appl 2013).

[0192] Notes:

[0193] Temperatures below 15° C. cause SDS precipitation and thus must be avoided during the sample processing steps. The lysate could be aliquoted and stored at −20° C. and, when needed, processed further after heating it up for 2 min at 95° C.

[0194] 2.2 Alkylation of Cysteine Residues

[0195] IAA stock solution is added to a final concentration of 150 mM with the incubation step being at least 15 min in a dark.

[0196] 2.3 Preparation of the Trypsin Solution

[0197] Trypsin solution (0.033 μg/μl in 50 mM NH.sub.4HCO.sub.3) is prepared prior to the step 2.4 and placed on ice.

[0198] 2.4 Sample Processing by STrap (Tryptic Digest and Peptide Desalting) [0199] 1. Pre-heat the heating block to 47° C. [0200] 2. Insert the S-tip into the 0-tube. [0201] 3. (See Notes) Add 120 μl of the STrapping buffer into the S-tip onto the top of the quartz stack. Wait for 1 min. [0202] 4. (See Notes) To 18 μl of the sample add 2 μl of the Phosphoric acid stock solution. Mix by pipetting up and down. [0203] 5. Slowly add the acidified sample into the upper quarter of the STrapping buffer in the S-tip. Insert the S-tip into the 0-tube. Place the Spin-unit into the centrifuge and mark the S-tip part facing outwards. [0204] 6. (See Notes) Centrifuge the Spin-unit at 2800×g for 2 min. [0205] 7. Dispose of the tube with the flow-through. [0206] 8. Add 70 μl of the STrapping buffer into S-tip. Insert the S-tip into the fresh 0-tube. Place the Spin-unit into the centrifuge with the S-tip mark facing inwards. Centrifuge the Spin-unit for 45 sec at 2800×g. [0207] 9. Add 30 μl of the AmBic solution into S-tip and centrifuge the Spin-unit for 30 sec at 2800×g. [0208] 10. Add 22 μL of the Trypsin solution into the S-tip onto the top of the plugs stack. Push down the solution using the syringe with a customized tip adapter till the solution meniscus is positioned ca. 3 mm above the top of the plugs stack. [0209] 11. Close the top of the S-tip with the 10 μl filter tip. [0210] 12. Insert the closed S-tip into the fresh 0-tube, place the unit into the heating block at 47° C. and cover with the aluminium foil. [0211] 13. (See Notes) Incubate for 60 min. [0212] 14. Remove the Spin-unit from the heating device. Take out the filter tip. Add 50 of the AmBic solution into the S-tip onto the top of the plugs stack. Wait 30 sec. [0213] 15. Centrifuge the Spin-unit at 2300×g for 60 sec. [0214] 16. (Optional) Remove the S-tip from the 0-tube. Add 3 μl of the TFA solution 2 to the flow-through. Load the acidified flow-through into the S-tip. Insert the S-tip back into the 0-tube. Centrifuge the Spin-unit at 2300×g for 60 sec. [0215] 17. Add 100 μl of the TFA solution 1 into the S-tip. Centrifuge the Spin-unit at 2500×g for 90 sec. [0216] 18. Place the S-tip into the fresh 0-tube. Add 80 μL of the Elution solution into the S-tip, centrifuge the Spin-unit for 5 sec at 2500×g, wait 30 sec and then centrifuge the Spin-unit for 1.0 min at 2500×g. [0217] 19. (Optional) Add 50 μl of the Elution solution into the S-tip, centrifuge the Spin-unit for 60 sec at 2500×g. [0218] 20. The eluate in the 0-tube, containing desalted peptides, is concentrated in the SpeedVac to the final volume of 5-12 μL. If needed, the concentrated peptide mixture could be diluted with the FA solution up to the required volume. To remove any particulate matter, spin down the peptide samples before loading them into the autosampler vials or plates.

Notes:

[0219] 1. After each centrifugation step make sure that all added solution has gone through the S-tip. [0220] 2. A properly assembled S-tip can tolerate the centrifugal acceleration of at least 4000×g. [0221] 3. The typical working ratio between the STrapping buffer in the S-tip (step 3) and the acidified sample (step 4) is 6:1 (acceptable tested ranges 4.5:1 to 7:1), e.g. the STrapping buffer 120 μl and the added acidified sample 20 μl. [0222] 4. In step 4, the sample could be diluted with the Lysis buffer up to the required volume before the acidification. [0223] 5. The final concentration of the phosphoric acid in the sample (step 4) is 1.2% which is achieved by addition of the phosphoric acid stock solution to the sample at 1:10 ratio. [0224] 6. Alternatively to the high-temperature (47° C.) digestion, digestion for 3-4 hours at 37° C. in a humidified chamber could be performed.

[0225] Cys-STrap Method

[0226] The following method is an adaptation of the method described above, where the surface of the matrix is modified to increase the affinity of the surface of the matrix for cysteine residues of protein/protein fragments specifically, thereby allowing those proteins/protein fragments that comprise cysteine residues to be analysed separately to those proteins/protein fragments that do not comprise cysteine residues.

[0227] Tryptic Digest with Enrichment of the Cysteine Containing Peptides, Maximum Protein Load 60 μg

[0228] 1. Materials

[0229] Solutions and Reagents [0230] Milli-Q water (H20) [0231] Methanol [0232] AmBic (Ammonium Bicarbonate) solution: 40 mM NH.sub.4HCO.sub.3 in H.sub.2O [0233] AmBic-ACN solution: 20% acetonitrile in 40 mM NH.sub.4HCO.sub.3 [0234] Lysis buffer: 5% (w/v) sodium dodecyl sulfate (SDS), 50 mM Tris/HCl pH 7.5 [0235] STrapping buffer: 90% methanol, 100 mM Tris/HCl pH 7.1 [0236] Phosphoric acid solution: 12.15% in H.sub.2O [0237] DTT solution: 1 M dithiothreitol (DTT) in H.sub.2O, freshly prepared [0238] DTT elution solution: 50 mM OTT, 5% acetonitrile, 40 mM NH.sub.4HCO.sub.3, freshly prepared [0239] IAA solution: 1 M iodoacetamide in H.sub.2O, freshly prepared [0240] Trypsin solution: 0.1 μg/μl of trypsin (V5111, Promega) in 40 mM NH.sub.4HCO.sub.3, prepared [0241] prior to starting the Cys-STrap processing step and placed on ice [0242] FA solution 1: 2% formic acid in H.sub.2O [0243] FA solution 2: 50% acetonitrile, 0.5% formic acid in H.sub.2O [0244] FA solution 3: 10% formic acid [0245] Isopropanol solution: 50% isopropanol in H.sub.2O

[0246] Equipment [0247] Bench-top centrifuge (for example MiniSpin, Eppendorf) [0248] Probe sonicator (for example Soniprep 150, MSE) [0249] Heating block suitable for handling 1.5 ml microtubes (for example PHMT, Grant Bio) [0250] Plastic syringe, 20 ml (for example 301031, BO) with a custom adapter to fit into 200 μl pipette tips

[0251] Vacuum concentrator (for example SpeedVac, Thermo)

[0252] Cys-STrap Tip

[0253] MK360 quartz filter is modified with pyridyldithiol, i.e. MK360-50 mm filter is incubated with 10 ml of 2% (3-Aminopropyl)triethoxysilane (APTES) soluton in acetone for 20 min and then washed several times with acetone. 5 mg of N-succinimidyl 3-(2-pyridyldithiol)propionate (SPDP) is dissolved in 0.4 ml of dimethyl sulfoxide (OMSO) and added into 9.6 ml of phosphate buffered saline (PBS) (pH 7.4, 15 mM EOTA). The aminopropyl-modified filter is incubated with the resultant SPDP solution for 2 hours at room temperature and then washed several times with the PBS/15 mM EDTA solution and air dried overnight. Using a 14 gauge blunt needle, the Cys-STrap tip is constructed by inserting 12 plugs of the pyridyldithiol modified MK360 material into a 200 μl pipette tip—similarly to the original STrap tip protocol however no underlying C.sub.18 membrane compartment is added in this case.

[0254] O-tube: A 1.5 ml microcentrifuge tube (72.690.001, Sarstedt) with an opening punctured in the tube's lid (alternatively, a pipette tip lid-adapter for microcentrifuge tubes could be used). The Cys-STrap tip and 0-tube comprise the Spin-unit

[0255] Filter tips, 10 μl (TF-300-R-S, Axygen)

[0256] 2. Methods

[0257] 2.1 Cell Lysis and Reduction of Cysteine Residues

[0258] Cells are lysed in excess of the Lysis buffer (ca. 1:10 sample-to-Lysis buffer volume ratios) at room temperature. To shear the DNA, the lysate is shortly sonicated using a probe sonicator. DTT solution is added to the final concentration of 20 mM. The extract is heated up at 95° C. for 5 min. The extract is clarified by centrifugation at 12,000×g for 10 min.

[0259] 2.2 Preparation of the Trypsin Solution

[0260] Trypsin solution (0.1 μg/μI in 40 mM NH.sub.4HCO.sub.3) is prepared prior to the step 2.3 and placed on ice.

[0261] 2.3 Sample Processing by Cys-STrap [0262] 1. Pre-heat the heating block to 47° C. [0263] 2. (See Notes) Add 120 μI of the STrapping buffer into the Cys-STrap tip onto the top of the quartz stack. Wait for 1 min. [0264] 3. (See Notes) To 18 μl of the sample add 2 μl of the Phosphoric acid stock solution. Mix by pipetting up and down. [0265] 4. Slowly add the acidified sample into the upper quarter of the STrapping buffer in the Cys-STrap tip. Insert the Cys-STrap tip into the 0-tube. Place the Spin-unit into the centrifuge and mark the Cys-STrap tip part facing outwards. [0266] 5. Centrifuge the Spin-unit at 2500×g for 1 min. [0267] 6. Dispose of the tube with the flow-through. [0268] 7. Add 70 μl of the STrapping buffer into Cys-STrap tip. Insert the S-tip into the fresh O-tube. Place the Spin-unit into the centrifuge with the Cys-STrap tip mark facing inwards. Centrifuge the Spin-unit for 45 sec at 2500×g. [0269] 8. Add 40 μI of the AmBic solution into Cys-STrap tip and centrifuge the Spin-unit for 30 sec at 2500×g. [0270] 9. Add 35 μL of the Trypsin solution into the Cys-STrap tip onto the top of the plug stack. Push down the solution using the syringe with a customized tip adapter till the solution meniscus is positioned ca. 4 mm above the top of the plug stack. [0271] 10. Close the top of the Cys-STrap tip with the 10 μl filter tip. [0272] 11. Insert the closed Cys-STrap tip into the fresh 0-tube, place the unit into the heating block at 47° C. and cover with the aluminium foil. Incubate for 60 min. [0273] 12. Remove the Spin-unit from the heating device. Take out the filter tip. Add 40 of the AmBic solution into the Cys-STrap tip onto the top of the plug stack. [0274] 13. Centrifuge the Spin-unit at 2000×g for 30 sec. [0275] 14. Add 50 μl of the AmBic-ACN solution into the Cys-STrap tip onto the top of the plug stack. Centrifuge the Spin-unit at 2000×g for 30 sec. [0276] 15. Collect the flow-through fraction. The peptides in this fraction could be either fractionated by ion-exchange (e.g. SAX STAGE tip fractionation.sup.1) or cleaned by the basic C.sub.18 STAGE tip.sup.2 method after having evaporated acetonitrile. [0277] 16. Cys-STrap tip is washed consecutively with 100 μl of the FA solution 1, FA solution 2, isopropanol solution, and Ambic-ACN solution using centrifugation in the Spin-unit and decanting the flow-through when necessary. [0278] 17. Cys-STrap tip is washed with 50 μl of methanol solution using centrifugation in the Spin-unit. [0279] 18. 40 μl of DTT elution solution is added into the Cys-STrap tip. The tip is placed into a fresh tube, the DTT elution solution is pushed down with the syringe-adapter till the solution meniscus is positioned ca. 5 mm above the top of the plug stack. Incubation is performed at 37° C. for 45 min. [0280] 19. 30 μl of the Ambic solution is added into the Cys-STrap tip and pushed down with the syringe-adapter. [0281] 20. 50 μl of the Ambic-ACN solution is added into the Cys-STrap tip and pushed down with the syringe-adapter. [0282] 21. 20 μl of the IAA solution is added to the eluate and the mixture is incubated in the dark for 30 min. [0283] 22. The mixture is acidified by the FA solution 3 to the final concentration of 0.5% formic acid. [0284] 23. Following the above alkylation step, the peptides could be cleaned up by the SCX tip extraction (e.g. the peptides could be loaded onto the SCX STAGE tip pre-activated with the consecutive washes of methanol and 20% acetonitrile in 0.5% formic acid, washed with 20% acetonitrile in 0.5% formic acid, eluted with 0.7 M Ammonium Acetate in 20% acetonitrile, dried down, reconstituted in 0.2% formic acid/2% acetonitrile for the consequent analysis by LC-MS).

Note:

[0285] 7. After each centrifugation step make sure that all added solution has gone through the Cys-STrap tip. [0286] 8. The typical working ratio between the STrapping buffer in the Cys-STrap tip and the acidified sample is 6:1.

REFERENCES

[0287] 1. Wisniewski, J. R., Zougman, A. & Mann, M. Combination of FASP and

[0288] StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res 8, 5674-5678, doi:10.1021/pr900748n (2009). [0289] 2. Rappsilber, J., Mann, M. & lshihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature protocols 2, 1896-1906, doi:10.1038/nprot.2007.261 (2007).

[0290] Targeted Peptide Enrichment with STrap

[0291] If the depth filter surface is silica-based, it is easily modifiable with functional groups based on the silane chemistry. Thus, during the digest, peptides possessing targeted features could be captured/enriched in the STrap unit. As an example, the quartz depth filter could be activated with the pyridyldithiol group (via consecutive reactions with (3-Aminopropyl)triethoxysilane and N-succinimidyl 3-(2-pyridyldithio)propionate) enabling the capture of the cysteine containing peptides. In this case, the modified STrap unit contains only the depth-filter part. The protein particulate is captured and digested in the depth filter. The cysteine-containing peptides are covalently attached to the quartz surface during the digest, the uncaptured peptides are eluted into the collecting tube for the downstream processing (e.g. further fractionation steps such as strong-anion exchange, reversed-phase clean up and downstream analysis), the STrap unit is washed rigorously and the cysteine-containing peptides are eluted using a reducing agent such as dithiothreitol, for example, alkylated and cleaned up for further analysis. An example of the output of the cysteine enrichment STrap (Cys-STrap) method applied to the bottom-up proteomics profiling of the MGH-U3 bladder cancer cell line according to the provided Cys-STrap protocol is presented in FIG. 11. We can observe the separation of the peptide products into two distinct populations—the flow-through (21509 peptides, 98% unique) and cysteine-enriched (6809 peptides, 95% unique, 93% cysteine-containing). The proposed concept is not limited to the cysteine peptide enrichment only and could be used with other molecular probes targeting either specific amino acids or amino acid modifications, e.g. the filter could be derivatized with (3-Glycidyloxypropyl)trimethoxysilane and then NHrmodified aptamer ligands could be covalently attached to the silica surface (e.g. such as the aptamers against L-Arginine1 to enrich for Arginine-containing peptides) or, by the same token, the filter could be functionalized with aminophenylboronic acid to enrich for glycopeptides.

REFERENCES

[0292] 1. Harada, K. & Frankel, A. 0. Identification of two novel arginine binding ONAs. The EMBO journal 14, 5798-5811 (1995). [0293] 2. Weith, H. L., Wiebers, J. L. & Gilham, P. T. Synthesis of cellulose derivatives containing the dihydroxyboryl group and a study of their capacity to form specific complexes with sugars and nucleic acid components. Biochemistry 9, 4396-4401 (1970).

[0294] Serum Processing with STrap

[0295] The application is based on the observation that in −2% reducing Sodium Oodecyl Sulfate (SOS) solution and upon acidification with phosphoric acid some abundant serum proteins, such as albumin, are precipitated out of the solution (FIG. 12), thus allowing direct proteomics analysis of the acidified SOS-solubilized serum samples based on the STrap principles (e.g. using the Cys-STrap method) without employing preliminary serum depletion procedures, which are typically performed in order to remove abundant serum proteins that severely interfere with identification of the low abundant proteins by mass spectrometry. Using the processing protocol outlined below in combination with the Cys-STrap method we have identified −370 proteins in a normal human serum sample, which is an increase of −2 times in protein identifications over a typical proteomics profiling of the undepleted serum.

[0296] Serum processing with modified STrap protocol (outline) [0297] 1. To 10 μl of serum add 10 μl of the reducing SDS solution (4.5% (w/v) sodium dodecyl sulfate, 50 mM Tris/HCl pH 7.5, 40 mM dithiothreitol) [0298] 2. Heat the sample for 7 min at 95° C. in the heating block [0299] 3. Remove the sample from the heating block and incubate for 20 min at room temperature (RT) [0300] 4. To the sample add 2.5 μl of 12.15% phosphoric acid [0301] 5. Centrifuge the sample at 12,000×g for 10 min [0302] 6. Carefully collect the supernatant for further processing with the Cys-STrap method Discussion of Depth Filters Relevant for the Present Invention

[0303] Depth filters have a random network of pore channels that vary in size and geometry. They are manufactured from a variety of solid materials. Materials of construction include various forms of plastics, cellulose, and glass, either singly or in combination. The processes used to manufacture depth filters do not result in a regular arrangement of the solid matrix. Instead, there is a range of pore sizes within a given structure that includes pores significantly larger and significantly smaller than the pore rating.

[0304] The randomness of the structure does not allow the assignment of a definitive upper limit on the size of particles that may pass through the filter. A portion of the particles in the filtrate will exceed the pore rating. Depth filters also can entrap a large percentage of particles smaller than the pore rating. Because depth filters trap particles throughout the structure, they typically exhibit a high particle-handling capacity. This makes them particularly useful in applications where the solution being filtered has a high particle load. Depth filters are not considered sterilizing-grade.

[0305] FIG. 9 shows the results of comparison of various grades of flat filters, i.e. Grade 4 (20-25 μm), Grade 598 (8-10 μm) and Grade 3 (6 μm). Ten microgram of Hela SDS lysate were acidified and precipitated in the neutral methanolic solution according to the STrap protocol and passed through the two layers of a filter paper stacked in a pipette tip. The trapped protein material was eluted with 2× sample loading buffer and visualized with the use of the Coomassie-stained polyacrylamide gel.

[0306] As can be seen, all three filters achieved some degree of retention, but the finer Grade 3 flat filter (6 μm cut-off) provided significantly improved performance and is able to retain about 50% of the loaded material. Thus the indication is that depth filters in the trapping range of from 10 μm down to 0.1 μm (or even smaller) are preferred, e.g. about 5 μm or finer, about 1 μm or finer, about 0.5 μm or finer being suitable.

[0307] Depth filters are typically used as pre-filters because they are an economical way to remove ≥98% of suspended solids and protect elements downstream from fouling or clogging. They owe their high capacity to the fact that contaminants are trapped and retained within the whole filter depth.

[0308] Conventional depth filters can be made out of the following materials: [0309] Quartz [0310] Glass Fibre [0311] Polymers [0312] Cellulose

[0313] Quartz

[0314] Filter media made of pure micro-quartz fibres. Such media can be produced with or without glass fibres and binder. Media without glass fibres and binder are particularly appropriate for emission control at high temperatures of 900-950° C. and wherever absolute purity of the filter medium is required. Excellent filtration properties, minimal metal contents, outstanding weight and dimension stability. Examples in include MK360 (Munktell), a preferred filter material for the present invention.

[0315] Glass Fibre

[0316] As implied by the name, glass fibre depth filters are made from glass fibres. In sheet form the fibres are initially held together only as a consequence of mechanical interaction. To improve the handling characteristics, the filter is sometimes treated with a polymeric binder, such as polyvinyl alcohol, which serves to hold the matrix together. Glass fibre filters are also prone to fibre shedding. If required, a membrane filter can be placed downstream to retain any fibres. Examples include GF/D (Whatman), a filter material which is utilised in the above-mentioned examples.

[0317] Polymers

[0318] Polymeric depth filters are manufactured from plastic fibres of various lengths, morphologies, and diameters. To improve the strength of these filters and reduce the level of fibre shedding, the filter can be calendered, the process of running the material between cylindrical rollers to apply pressure and/or heat. Most polymeric depth filters are inherently hydrophobic. For low pressure aqueous filtration, the filter may require a surface treatment to render it wettable. Polymeric depth filters are normally very strong and easy to handle.

[0319] Cellulose

[0320] As implied by the name, cellulosic depth filters are made from cellulose fibres. The fibres can be derived from a relatively crude source, such as wood pulp, or a highly purified source, such as cotton. The filters are manufactured by techniques very similar to paper manufacture and are very economical. Although they are generally very easy to handle when dry, they are mechanically very weak when wet. Cellulosic filters are prone to fibre shedding during fabrication into a device and when used in filtration. If required, a membrane filter can be placed downstream to retain any fibres. Typically cellulose fibres are less preferred for the present invention, primarily due to the potential for contaminants and possible reaction with the proteins of the sample. However, certain highly purified forms may well be useful.

REFERENCES

[0321] 1. Zhang, Y., Fonslow, B. R., Shan, B., Baek, M. C. & Yates, J. R., 3rd Protein Analysis by Shotgun/Bottom-up Proteomics. Chem Rev 113, 2343-2394 (2013). [0322] 2. Yu, Y. Q., Gilar, M., Lee, P. J., Bouvier, E. S. & Gebler, J. C. Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal Chem 75, 6023-6028 (2003). [0323] 3. Kadiyala, C. S., Tomechko, S. E. & Miyagi, M. Perfluorooctanoic acid for shotgun proteomics. PLoS One 5, e15332 (2010). [0324] 4. Summers, D. F., Maizel, J. V., Jr. & Darnell, J. E., Jr. Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc Natl Acad Sci USA 54, 505-513 (1965). [0325] 5. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970). [0326] 6. Shevchenko, A., Wilm, M., Vorm, 0. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-858 (1996). [0327] 7. Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat Methods 6, 359-362 (2009). [0328] 8. Havlis, J., Thomas, H., Sebela, M. & Shevchenko, A. Fast-response proteomics by accelerated in-gel digestion of proteins. Anal Chem 75, 1300-1306 (2003). [0329] 9. Zhou, H., Ning, Z., Wang, F., Seebun, D. & Figeys, D. Proteomic reactors and their applications in biology. Febs J 278, 3796-3806 (2011). [0330] 10. Gilar, M., Olivova, P., Daly, A. E. & Gebler, J. C. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J Sep Sci 28, 1694-1703 (2005). [0331] 11. Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mo/Syst Biol 7, 548 (2011). [0332] 12. Wisniewski, J. R. & Mann, M. Spin filter-based sample preparation for shotgun proteomics Reply. Nat Methods 6, 785-786 (2009). [0333] 13. Loyer, P. et al. Characterization of cyclin L 1 and L2 interactions with CDK11 and splicing factors: influence of cyclin L isoforms on splice site selection. J Biol Chem 283, 7721-7732 (2008). [0334] 14. Bordier, C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256, 1604-1607 (1981). [0335] 15. Wisniewski, J. R., Dus, K. & Mann, M. Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins. Proteomics Clin Appl 7, 225-233 (2013). [0336] 16. Di Palma, S., Mohammed, S. & Heck, A. J. ZIC-cHILIC as a fractionation method for sensitive and powerful shotgun proteomics. Nat Protoc 7, 2041-2055 (2012). [0337] 17. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367-1372 (2008). [0338] 18. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10, 1794-1805 (2011).