Method of detection of amino acid sequence and/or identification of peptides and proteins, by use of a new derivatization reagent and synthesis of 5-formyl-benzene-1,3-disulphonic acid as derivatization reagent

Abstract

Present invention refers to a novel and improved method of derivatization and detection of amino acid sequence and/or identification of proteins, peptides by a new derivatization compound. Precisely, the method discloses a novel approach to derivatization of peptides or proteins by compounds comprising two or more sulfonyl groups and analysis of derivatized analytes in negative mode of operation of mass spectrometer. This method allows unambiguous analysis of amino acid sequence of long-chain peptides/proteins. Also, the invention discloses a novel synthesis procedure of 5-formyl-benzene-1,3-disulphonic acid as derivatization compound.

Claims

1. A method of detection of amino acid sequence and identification of peptides and/or proteins, comprising the following steps: selective derivatization of peptides and/or proteins at N-terminus having unprotected epsilon-amine of lysine, wherein the selective derivatization is carried out at pH 1-5 with addition of sodium cyanoborohydride (NaBH.sub.4CN) using 5-formylbenzene-1,3-disulphonic acid, wherein the selective derivatization comprises reacting of the aldehyde group with an amino group at the N-terminus of the peptide or protein to form derivatized analytes in a form of a secondary amine; analysis of one or more of the derivatized analytes by acquisition of spectra of derivatized negative ions acquired in the MS/MS negative mode of operation of a mass spectrometer; and interpretation of an obtained fragmentation pattern to detect amino acid sequence and to identify the aforementioned analyte.

2. The method according to claim 1, wherein in the analysis step, the spectra of derivatized negative b-ions is acquired in the MS/MS negative mode of operation of the mass spectrometer.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 represents the route of synthesis of 5-formyl-benzene-1,3-disulphonic acid (synthesis is described in Detailed description of the invention);

(2) FIG. 2 represents cleavage mechanism of analytes in mass spectrometer;

(3) FIG. 3 represents MS/MS mass spectra for equal amounts of analyzed analytes of tryptic digests of trypsin autolysis (1 ng) of: (3A) LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys peptide SEQ ID NO: 1) derivatized by derivatization compound 4-formyl-benzene-1,3-disulphonic acid with two sulfonyl groups in ortho and para position acquired in the negative mode of operation of mass spectrometer and (3B) LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys peptide SEQ ID NO: 1) derivatized with derivatization compound 5-formyl-benzene-1,3-disulphonic acid with two sulfonyl groups in meta and meta position acquired in negative mode of operation of mass spectrometer.

DETAILED DESCRIPTION OF THE INVENTION

Analysis of Peptides/Proteins Using the Compound of General Formula I

(4) The method presented by the subject invention enables detection of complete amino acid sequence of proteins, i.e., peptides. In protein identification, protein cleavage with known chemical or enzymatic proteolytic procedures precedes the method of the subject invention. Thus, proteins can be cleaved for example with chemical compounds such as ninhydrin, cyanogen bromide or by simple degradation using acid hydrolysis. Furthermore, proteins can be cleaved with enzymes such as trypsin, chymotrypsin, thermolysin, Lys-C, Glu-C, Arg-C, etc.

(5) The procedure of protein proteolysis, involving in the majority of cases peptides up to 5000 Da, with the possibility of derivatization of larger peptides as well, is followed by peptide mass determination by mass spectrometry and thereafter peptide derivatization procedure. Derivatization compound of general formula I is added to aqueous solution of peptides/proteins to be derivatized. Derivatization compound binds to N-terminus of peptide via reactive group giving it two sulfonyl groups and increasing its mass. Therefore, during the mass spectrometry, it is necessary to add thereto the m/z value of the relevant derivatization compound comprising two sulfonyl groups to the peptide mass (in MS/MS positive mode of operation the reagent is detached during analyses yielding products; y-ions that can reveal amino acid sequence, whereas in MS/MS negative mode of operation the reagent is not detached from N-terminus, but the formation of products; derivatized b-ions revealing amino acid sequence is still increased). Therefore, N-terminus is doubly negatively charged, whereas C-terminus or some basic side branch of peptide and/or protein is positively charged. When sulfonyl groups donate labile protons to amide groups, peptide and/or protein dissociates into amino acid integral portions. Resulting positive ions do not differ in mass from the positive ions of non-derivatized analogue, since sulfonyl groups during proton donation to amide bond in the peptide cleave off either a portion of ions from N-terminus of peptide chain or derivatization compound itself, thus producing gradually shorter peptide/protein ions. The same mechanisms in negative mode of operation yields the final reaction products, i.e., negatively charged derivatized ions comprising two sulfonyl groups at N-terminus, which increase the mass of precursor ions and product ions for the mass values of derivatization reagent (FIG. 2). However, regardless of the addition of derivatization group, the differences between detected ions in positive or negative mode of operation of mass spectrometry are equal, which ultimately enables determination of amino acid sequence or their modifications. The arrangement of sulfonyl groups with respect to the site where aldehyde of derivatization compound binds with peptide amine is equidistant (meta, meta), i.e., both groups are equally distant from the binding site, but they are not in direct proximity of reactive group. Such arrangement of sulfonyl groups with respect to the other disulfonyl analogues allows easier proton donation to amide groups on the peptide.

(6) Derivatized negative ions can be analysed in different mass spectrometers. Thus, the following mass spectrometers are used: time-of-flight (TOF), tandem mass analyzers (MS/MS, QQQ, MS/Q, Q/TOF), quadropole (Q), ion trap (IT) and similar devices. Moreover, experiments have shown that the use of MALDI ion source in the subject invention gives rise to exclusively y fragments of positive ions and derivatized negative b-ions. Signals obtained by analysis of these derivatized negative b-ions produced in the subject procedure are extremely intensive, whereas noise in comparison with standard methods of detection of amino acid sequence or identification of peptide/protein is up to 10-fold lower. Thus, the overall increase of signal intensity obtained by the subject invention is up to 15-fold higher than the signal intensities obtained by the methods in the state of the art, except in the case of derivatization by other derivatization compounds where sulfonyl groups are in ortho, para position with respect to the reactive group.

(7) In the particular example compared results are obtained by derivatization with the use of 4-formyl-benzene-1,3-disulphonic acid (compound of patent application P20100044A) and 5-formyl-benzene-1,3-disulphonic acid. In comparison with derivatization with the use of 4-formyl-benzene-1,3-disulphonic acid the improvement of signal intensity by amino acids which are 10 and more amino acids away from the site of derivatization is up to 10-fold higher with respect to the noise after peptide derivatization with 5-formyl-benzene-1,3-disulphonic acid (FIG. 3A and FIG. 3B and Table 1). Improvement of mobile proton range is allowed by different arrangement of sulfonyl groups (meta, meta in 5-formyl-benzene-1,3-disulphonic acid with respect to ortho, para in 4-formyl-benzene-1,3-disulphonic acid). Ortho sulfonyl group donates the majority of its protons to the nearest amine produced after derivatization of N-terminus, which is visible in spectra of peptide cleavage in negative mode of operation of mass spectrometer where measured signal at m/z 249 is often the most intensive in the spectrum (signal produced after reagent cleavage at the site of derivatization). Such pronounced derivatization reagent cleaving off is absent when sulfonyl groups are in meta, meta position. Moreover, meta, meta sulfonyl groups do not hinder derivatization of N-terminus as noticed when sulfonyl groups are in direct proximity of benzaldehyde group (ortho position). Due to said reasons ortho sulfonyl group hinders derivatization and uses the majority of its protons to cleave off the nearest nitrogen reducing the activity range of mobile proton towards distant amino acids. The comparison was carried out under the same conditions in mass spectrometer and with the same amount of derivatization peptides (1 ng of tryptic digest of trypsin autolysis). However, by using mass spectrometers that cleave peptides/proteins in a different manner, it is possible to obtain with the subject invention also other fragments of negative and positive ions, maintaining the basic component of amino acid sequence determination based on the difference between masses of signal sequence.

(8) Direct comparison of the compound used in the state of the art (4-formylbenzene-1,3-disulphonic acid) and the newly synthesized compound (5-formylbenzene-1,3-disulphonic acid) reveals increase of signal intensity of b-ions of amino acids, and extended range of derivatization reagent. This increase is most contributed by meta, meta arrangement of sulfonyl groups which do not sterically hinder the derivatization procedure, and do not enter into interaction in mass spectrometer with the nearest amino or amide protons.

(9) TABLE-US-00001 TABLE 1 Comparison of signal intensity of peptide b-ions 10 or more amino acids away from the site of derivatization of LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys peptide (SEQ ID NO: 1) after derivatization with 4-formyl-benzene-1,3- disulphonic acid (ortho, para) and with 5-formyl-benzene-1,3-disulphonic acid (meta, meta). Signals of b-ions less than 10 amino acids away were not taken into calculation considering that in that field calculated S/N is the same for both derivatization reagents. Distance from the Measured signal-to- Fragment Amino site of noise ratio (S/N) m/z acid derivatization (ortho, para)/(meta, meta) 1368 Glu 10 98, 5/533, 6 1425 Gly 11 20, 1/86, 8 1539 Asn 12 32, 0/140, 2 1668 Glu 13 23, 4/150, 7 1796 Gln 14 8, 4/107, 8 1943 Phe 15 8, 0/53, 9 2056 Ile 16 8, 9/101, 7 2170 Asn 17 8, 7/51, 2 2241 Ala 18 0, 0/15, 5

(10) Reactive group of derivatization compound can be any group reacting with amino group. It is preferable that reactive group of the compound used in derivatization step of the subject invention is selected from the group comprising aldehyde, keto-, isothiocyanate-, isocyanate-group, NHS ester, anhydride or activated carboxylic acid group.

(11) It is most preferred that, in the subject invention, reactive group of derivatization compound is aldehyde group. In the cases where in the subject invention compound with aldehyde reactive group at ph 1-5 is used, aldehyde group selectively reacts with N-terminus of peptide or protein, without binding to other amino groups in protein or peptide chain. By using this approach dual derivatization of tryptic peptides is avoided, since derivatization reaction performed according to the subject invention selectively derivatized N-terminus without the need of adding protective group at lysine. Since in this case lysine guanidination is redundant, the method of detection of amino acid sequence, i.e., peptide/protein identification is additionally simplified. Also, since guanidination, which causes significant quantitative losses during sample handling, is not needed, signal intensity in the analysis of derivatized negative ions in negative mode of operation of mass spectrometry is increased. It is most preferred that compound with aldehyde group in derivatization step is used at approximately pH 4. Furthermore, it is preferable that during the use of derivatization compound with aldehyde reactive group, NaCNBH4 is also added to aqueous solution as to reduce imino-group of the resulting Schiff base.

(12) Moreover, it is most preferred to use 5-formyl-benzene-1,3-disulphonic acid as compound in derivatization step.

One of the Embodiments of the Invention

Example 1

Use of 5-formylbenzene-1,3-disulphonic acid as a Derivatization Compound

(13) In this example the method of peptide derivatization of the subject invention by chemical reaction in two stages was used: first stage includes condensation of aldehyde and primary amine with production of Schiff base, and the second stage includes reduction of imines of Schiff base into amines. The reagents used included: 5-formyl-benzene-1,3-disulphonic acid (synthesized at Rudjer Boskovic Institute), NaCNBH4 (Merck, Darmstadt, Germany), and peptides obtained by trypsin autolysis (Merck, Darmstadt, Germany), CHCA matrix (-cyano-4-hydroxycinnamic acid, Sigma Aldrich, St. Louis, Wis., USA).

(14) Table 2 presents trypsin peptide and its ion detected by mass spectrometry, which are produced during trypsin autolysis prior to derivatization by 5-formyl-benzene-1,3-disulphonic acid.

(15) TABLE-US-00002 TABLE2 Peptideproducedbytrypsinautolysiswith theoreticalcalculationofmassesof correspondingions. FRAGMENT m+ H sequence(SEQIDNO:1) T3 2211.1000 LeuGlyGluHisAsnIleAspValLeuGlu GlyAsnGluGlnPheIleAsnAlaAlaLys

(16) Upon purification of 1 g fragments derived from trypsin autolysis using ZipTip technique, peptide mixture was evaporated using SpeedVac (Eppendorf, Germany) concentrator followed by addition of 1 mg 5-formyl-benzene-1,3-disulphonic acid and 4 mg NaCNBH.sub.4 dissolved in 100 l phosphate buffer pH 4.0 to dried concentrate. The solution was stored in the fridge at temperature of 4-8 C. for 12 hours to react. Subsequently, 10 l of the solution was purified again using ZipTip, dried in SpeedVac concentrator and dissolved in 5 l CHCA matrix with 5 mg/ml concentration. 1 l of this solution was applied onto MALDI metal plate and analysed by mass spectrometer.

(17) Derivatization procedure of peptides (SEQ ID NO: 1) obtained by protein cleavage by trypsin at N-terminus is illustrated by the following reaction:

(18) ##STR00003##

(19) The example of instrumental MS/MS fragmentation of analytes originally derived from trypsin autolysis shows significant improvement of structural analysis of produced analytes, which increases accuracy of amino acid sequence detection (so caIled de novo sequencing of unknown peptides).

(20) For the purpose of evaluation of method success, MS/MS spectra were acquired on MALDI-TOF/TOF instrument before and after derivatization. An example presented in FIG. 3 gives a comparison of MS/MS spectra of negative b-ions of ion derivatized by 4-formylbenzene-1,3-disulphonic acid of lysine fragment LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys (SEQ ID NO: 1) (FIG. 3A) and MS/MS spectra of negative b-ions after derivatization by 5-formyl-benzene-1,3-disulphonic acid (FIG. 3B). It is important to point out that MS/MS spectra of negative derivatized ions are acquired on the same sample, i.e., on the same ions obtained after derivatization, and that the amount of used analyte was the same as the conditions of acquisition of both derivatized peptides.

(21) Produced negative b ions differ in mass for m/z 247,945 in comparison with underivatized fragments. During MS/MS analysis of negatively charged ions of derivatized fragment, it is possible to detect only b ions that kept sulfonyl groups, which requires that an increment of m/z 247,945 is added (FIGS. 2A and 2B). Extremely intensive signal produced by cleaving off derivatization reagent m/z 249 (247,945+1H.sup.+) is absent in the case of use of 5-formyl-benzene-1,3-disulphonic acid which proved higher mobile proton affinity for distant amino acids (FIG. 3B). Extremely intensive signal m/z 249 indicates consumption of large portion of protons situated in ortho position in sulfonyl group on cleavage of the nearest amino group (site of derivatization, i.e., derivatization reagent).

(22) Comparison of spectra in FIGS. 3A and 3B shows significant increase in measured ion signals in spectrum in FIG. 3B in comparison with equivalent signals in FIG. 3A (signals ten and more amide bonds away from the site of derivatization), which proves that the subject method increases success in detection of amino acid sequence of peptides.

(23) If up to 10-fold lower noise detected in analysis of derivatized ions with 5-formylbenzene-1,3-disulphonic acid is added, the overall increase in signal-to-noise ratio (S/N) after derivatization is 15-fold for MS/MS negative ions. If ions of amino acids presented in FIGS. 3A and 3B are compared, significant difference is noticeable after the tenth signal in amino acid sequence (m/z 1368) in favour of signal increase of peptide ions derivatized with 5-formyl-benzene-1,3-disulphonic acid (Table 1 and FIG. 3B). Similar experiments were carried out on all ions consisting of 10 or more amino acids presented in Table 1 before and after derivatization, and obtained results do not differ from the results set out in this example.

(24) Method described in the subject patent application is technically rapid, cost effective and reliable, and could be of high value in proteomics analyses of various samples, especially the biological ones. In fact, only in humans the size of the whole proteome is estimated to several million protein species, and databases currently provide information on completely determined amino acid sequences for approximately 1.5 million proteins. The described method could, thus, facilitate simple determination of amino acid sequences of human proteins unidentified so far with significant medical implications (e.g. biomarker discovery). It would also be as simple to identify proteins for different biological species for which publicly accessible protein databases, such as NCBI and UNIPROT, contain no exact information on amino acid sequences.

(25) Subject method reduces the time of sub-structural analyses of proteins and peptides by mass spectrometry (MS/MS analysis) and increases accuracy when searching protein databases.

Detailed Description of 5-formyl-benzene-1,3-disulphonic acid Synthesis

(26) The numbers of synthesized intermediates and final product correspond to the numbers in FIG. 1.

1. O-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate) (2)

(27) To the solution of 3,5-dihydroxybenzaldehyde (630 mg, 4.5 mmol, Sigma-Aldrich, St. Louis, Wis., USA) 3.0 g (22 mmol) K.sub.2CO.sub.3 (Kemika, Zagreb, Croatia) and 1.25 g (10 mmol) N,N-dimethylcarbamoylchloride (Sigma-Aldrich, St. Louis, Wis., USA) were added in 50 ml dry acetonitrile. Reaction mixture was mixed for 24 hours at room temperature, 20 ml water and 0.5 g KOH were added and mixed another 30 minutes. The majority of acetonitrile was evaporated and water solution extracted with use of dichlormethane (30+20 ml). Organic extracts were washed with saturated NaCl solution, dried and evaporated. Pre-crystallization from methanol produced 980 mg (70%) of pure product.

(28) 1H NMR (CDCl.sub.3): 3.37 (s, 6H); 3.46 (s, 6H); 7.14 (t, J=2.3 Hz, 1H); 7.49 (d, J=2.3 Hz, 2H); 9.98 (s, 1H) ppm.

(29) 13C NMR (CDCl.sub.3): 38.90; 43.39; 121.33; 124.30; 137.53; 154.55; 186.77; 190.08.

2. S-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate) (3)

(30) Carbamate 2 (350 mg, mmol) was heated in 2 ml diphenyl ether (Sigma-Aldrich, St. Louis, Wis., USA) at 230 C. for 2 hours. With use of chromatography on silica gel column (Sigma-Aldrich, St. Louis, Wis., USA) in addition to hexane-dichlormethane-ethylacetate, 315 mg (90%) of pure product was obtained.

(31) 1H NMR (CDCl.sub.3): 3.04 (brs, 6H); 3.08 (brs, 12H); 7.88 (t, J=1.6 Hz, 1H); 8.00 (d, J=1.6 Hz, 2H); 9.99 (s, 1H) ppm.

(32) 13C NMR (CDCl.sub.3): 37.08; 131.16; 136.97; 137.25; 147.39; 165.53; 190.55 ppm.

3. S-benzylalcohol-3,5-bis(N,N-dimethylthiocarbamate) (4)

(33) Thylthiocarbamate 3 (315 mg) was dissolved in 40 ml ethanol and 50 mg NaBH.sub.4 (Sigma-Aldrich, Buchs, Switzerland) was added. The solution was mixed for 1 hour at room temperature, hydride residue was destroyed with ammonium chloride solution (Kemika, Zagreb, Croatia), ethanol was evaporated, and the product extracted with use of dichloromethane. Pure product in the amount of 320 mg (100%) was obtained.

4. S-benzylacetate-3,5-bis(N,N-dimethylthiocarbamate) (5)

(34) To the solution of compound 4 (320 mg) 1.0 ml triethylamine (Sigma-Aldrich, St. Louis, Wis., USA) and 0.9 ml acetic acid anhydride (Kemika, Zagreb, Croatia) were added in 30 ml dichloromethane. After 3 hours anhydride residue was destroyed with addition of methanol. Reaction solution was washed with 2% HCl solution, dried and evaporated. Acetate 5 in the amount of 330 mg (91%) was obtained.

(35) 1H NMR (CDCl.sub.3): 2.09 (s, 3H); 3.05 (brs, 12H); 7.51 (d, J=1.2 Hz, 2H); 7.60 (t, J=1.2 Hz, 1H) ppm.

(36) 13C NMR (CDCl.sub.3): 20.97; 36.98; 65.26; 129.77; 135.83; 137.15; 141.87; 166.15; 170.69 ppm.

5. Benzylalcohol-3,5-disulphonic acid (6)

(37) To the cooled mixture of formic acid (15 g, Kemika, Zagreb, Croatia), water (1.8 g) and hydrogen peroxide (30%, 1.5 g, Kemika, Zagreb, Croatia) 125 mg acetate 5 was added. Reaction mixture was mixed for 20 hours at room temperature and evaporated to dryness. The raw product was passed through a column of Amberlite IR-120 (H.sup.+), (Rohm and Haas, Philadelphia, Pa., USA); the water solution was evaporated to dryness, and the residue was dissolved in 20 ml methanol and heated to boiling point for 30 minutes. Evaporation of the solution produced 90 mg (95%) of product 6.

(38) 1H NMR (CD.sub.3OD): 4.68 (s, 2H); 7.91 (s, 2H); 8.21 (s, 1H) ppm.

(39) 13C NMR (CD.sub.3OD): 62.94; 122.09; 125.53; 142.91; 145.08 ppm.

(40) No alternative oxidation route was found in the literature, and there is the possibility that other peroxyacids in aqueous acid conditions are able to oxidize the resulting compound in the same manner.

6. Benzaldehyde-3,5-disulphonic acid (7)

(41) Alcohol 6 (100 mg, 0.37 mmol) was dissolved in 30 ml dry acetonitrile. Pyridinium chlorochromate (Prepared according to: E. J. Corey and W. Suggs, Tetrahedron Lett. 1975, 16, 2647-2650) in the amount of 90 mg (0.41 mmol) was added to the solution and mixed for 2 hours at room temperature. The solvent was evaporated and 50 ml methanol and 3 ml ammonia (25%) were added. After 3 hours the residue was filtered, the solvent was evaporated, and the remaining was passed through a column of Amberlite IR-120 (H.sup.+). Disulphonic acid 7 in the amount of 80 mg (80%) was obtained.

(42) There are many other, more or less alternative reagents for which authors claim that they oxidise alcohol into aldehyde.

(43) 1H NMR (D.sub.2O): 8.36 (s, 3H); 9.96 (s, 1H) ppm.

(44) 13C NMR (D.sub.2O): 126.65; 128.25; 129.05; 137.07; 193.86 ppm.

(45) Shown examples of the methods of derivatization and synthesis of 5-formyl-benzene-1,3-disulphonic acid present only some of the embodiments of the subject invention and have no limitation on the scope of patent protection of the subject invention.