CN101042377A - Method and reagent kit for enriching and sequencing peptide fragment containing histidine - Google Patents
Method and reagent kit for enriching and sequencing peptide fragment containing histidine Download PDFInfo
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Abstract
This invention provides one new method and agent case for collecting and testing group aminoacidemia peptides, which comprises the following steps: protein restores alkyl base and tryptone protein enzyme cut, peptides section N end amidogen decoration and group aminoacidemia peptides color spectrum collection and mass spectrum testing sequence; identifying the protein with peptides sections in mixture objects.
Description
Technical field
The present invention relates to a kind of sequence measurement and kit of protein; Specifically, the present invention relates to a kind of chromatograph enrichment of peptide fragment containing histidine and the method and the kit of mass spectrum order-checking.
Background technology
Along with the development of mass-spectrometric technique, biological mass spectrometry is being brought into play more and more important effect in the order-checking of protein and in identifying.But because protein molecule is too big, existing mass spectrometer is difficult to identify agnoprotein matter by whole protein molecule is directly checked order, but elder generation adopts chemical method or enzyme process that protein molecule is cut into less peptide section, the peptide section is checked order again.Because protein group contains a variety of protein, system is very complicated again, must separate before mass spectrophotometry, to reduce the complexity of system.The difference of the precedence of cutting according to Separation of Proteins in the experimental implementation or enzyme, current proteomic techniques strategy can rough segmentation be " (Top-down) from top to bottom " and " (Bottom-up) from top to bottom " two big classes.The former earlier carries out multidimensional to complicated protein mixture to separate, and carries out mass spectrophotometry after the more single protein that obtains or simple mixtures enzyme being cut; The latter carries out holoenzyme to complicated protein mixture earlier to cut, and peptide section potpourri is carried out carrying out mass spectrophotometry again after multidimensional is separated.Because there are defective in isolation technics itself or separation with being connected of follow-up evaluation, the resolution and the recovery as the protein chromatographic resolution are not high, two dimensional electrophoresis is unsuitable for separating protein strong-hydrophobicity or extreme isoelectric point and molecular weight, and being difficult for realizing that automation of operation reaches and mass spectral on-line coupling, application of " from top to bottom " strategy is subjected to a lot of restrictions.So " from top to bottom " strategy is more and more paid attention to, obtain application more and more widely.
" from top to bottom " Ce Lve advantage is that the peptide segment molecule is little, being beneficial to chromatographic resolution reaches and mass spectral on-line coupling, but it is too complicated that shortcoming is a peptide section potpourri, separation efficiency to chromatogram has proposed very big challenge, and the peptide section of the peptide section of high-abundance proteins matter and low abundance proteins may co-elute, thereby influences the evaluation of low abundance proteins.Therefore, reducing the complexity of peptide section potpourri, improve separation efficiency, reduce the co-elute of height abundance peptide section, is the key of this technical tactic.Because the existence of Protein Data Bank and labelled peptide (signature peptide) does not need all peptide sections of protein are identified, as long as and identify one or a few labelled peptide, just can identify whole protein molecule.This just for adopting chemical modification ancillary technique or the representative labelled peptide of affinity chromatography technology separation and concentration, realizes the analysis and the evaluation of whole protein group are provided the foundation by simplifying the sample system.
Histidine is the relatively low seed amino acid of abundance in the protein molecule, and the peptide section proportion in all peptide sections that contains histidine is lower, and most protein all contains histidine peptide section.According to statistics, 11,936 protein in 46.0 editions human protein's databases of Swiss Prot are cut through theoretical enzyme can produce 694,584 peptide sections, wherein has only 127,143 (accounting for 18.3%) to contain the histidine peptide, but can cover 97.1% protein.Therefore, enrichment histidine peptide section can reduce the complexity of peptide section potpourri greatly.Basic solid metallic affinity chromatography (IMAC) technology that adopts is come enrichment histidine peptide in the document
[1-4]Regnier etc. investigate selectivity, the recovery, reappearance, specificity and the column life of this method, find that its selectivity, the recovery and reappearance are subjected to the influence of elution requirement very big, capture rate to histidine peptide in the standard protein is 85%, also observes losing of non-specific bond and metallic ion
[5]The more important thing is that these are without the cleavage of mass spectrum behavior more complicated of the peptide section of chemical modification, influenced the quality of its second order ms figure and the analysis of its sequence.Bibliographical information is arranged
[6-7]Cut the N end of peptide section at the pancreatin enzyme and introduce sulfonic group, can promote the cracking of peptide section and simplify its second order ms figure, make the continuous y ion of appearance among its second order ms figure, make the analysis of the parsing of second order ms figure and peptide sequence very simple, also improved the confidence level of identification of proteins.
Summary of the invention
The invention provides a kind of simple effectively, but both enrichment contained the histidine peptide, can promote its mass spectrum order-checking again, improved the method for identification of proteins confidence level.This method may further comprise the steps:
(1) opens disulfide bond in the protein molecule with reductive agent, destroy its higher structure; Seal sulfydryl with alkylating reagent, prevent that it from forming disulfide bond again;
(2) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry; Protect with the side chain amino that reagent is cut the peptide section to enzyme;
(3) the peptide section is carried out chemical modification and processing and carried out mass spectrophotometry;
The disposal route that it is characterized in that described step (3) is:
(a) use one or more chemical reagent, the N end amino of peptide section is modified, make it be with acidic-group;
(b) peptide section sample is carried out desalination;
(c) contain the histidine peptide with cation-exchange chromatography post separation and concentration;
(d) make the mass spectrogram that contains histidine peptide cracking generation fragmention with mass spectrum, mass spectrometric data is resolved, identify to produce the protein that contains the histidine peptide.
Wherein, acidic-group is selected from one or more in the following sulfonic acid described in the step (a): 2-sulfo group acetyl group, 3-sulfo group propiono, 2-sulfo group benzoyl or 3-sulfo group benzoyl.
The method of desalination is a reversed phase chromatography described in the step (b), preferred on-line coupling reversed phase chromatography.
Mass spectrum described in the step (d) is selected from MALDI tandem mass spectrum, electro-spray ionization tandem mass spectrum or liquid one matter coupling tandem mass spectrum, preferred MALDI tandem mass spectrum; Preferred commodity in use software of the parsing of described mass spectrometric data and the authentication method of protein or database search.
The N end that this method is cut the peptide section at the protein enzyme adds stronger acidic-group, contains the histidine peptide to reduce the complexity of peptide section potpourri by the ion-exchange chromatography separation and concentration, carries out mass spectrophotometry then to identify crude protein.The acidic-group that is added in the N end is electronegative under the ion-exchange chromatography condition, neutralized peptide section C end alkaline amino acid residue with positive charge, making the non-histidine net charge that peptide is with that is in the great majority is zero, and account for fraction contain the histidine peptide still with positive net charge, thereby be implemented in separation and the enrichment that contains the histidine peptide in the ion-exchange chromatography.In mass spectrophotometry, C end alkaline amino acid residue is by protonated the time, the acidic-group of N end is by deprotonation, both electric charges neutralize mutually, what have the alkalescent amino acid residue contains the histidine peptide with respect to non-histidine peptide, in the positive ion analytical model, be easier to catch proton and be ionized, thereby have higher Ionization Efficiency and detection sensitivity.And another proton that the more weak histidine residues of alkalescence does not influence this peptide section of ionization moves and amide group protonated at random along " freedom " of peptide backbone.The present invention is achieved by above-mentioned principle.
Utilize this method, greatly reduce the complexity of peptide section potpourri, significantly improved and contained chance that the histidine peptide detected by mass spectrum and the lysis efficiency in second order ms thereof, the signal to noise ratio (S/N ratio) of fragmention is improved greatly, the quality of second order ms figure is obviously improved, and can realize containing the de novo sequencing of histidine peptide according to second order ms figure.
On the other hand, the present invention also provides a kind of kit that is used for peptide fragment containing histidine enrichment and order-checking, and the component of this kit comprises:
(a) one or more N end amino to the peptide section are modified, and make it with the reagent of going up acidic-group;
(b) one or more can carry out the equipment that the desalination of peptide section separates with cation-exchange chromatography;
(c) one or more are used for the buffer system that the desalination of peptide section separates with cation-exchange chromatography.
Wherein, the preferred sulfonic acylating reagent of tool of component (a); The conventional analysis chromatographic column or the rifle hair style of the preferred on-line coupling type of component (b) are filled pillar.
Use for convenient, this kit also can comprise one or more in the following component: be used for opening the reductive agent of protein molecule disulfide bond, as dithiothreitol (DTT) (DTT) and three carboxyethyl phosphines (TCEP); The sealing sulfydryl prevents that it from forming the alkylating reagent of disulfide bond again, as iodoacetamide and iodoacetic acid; The protein digestibility agent is as trypsase; The side chain amido protecting agent is as the O-methyl-isourea; Can contain one or more digestion assistants in addition; The operation instruction of state administrative organs's approval etc.
New method of the present invention and kit are applicable to the enrichment and the mass spectrum order-checking of peptide fragment containing histidine protein in protein or the mixtures of polypeptides, are specially adapted to carry out in biology, medical science and the field of pharmacology protein or Proteomic analysis.Method of the present invention and kit have practicality widely.Purposes includes but not limited to: the proteome research that carries out various animals and plants and microorganism in fields such as medical science, pharmacy, agricultural and animal husbandry.
Description of drawings
Fig. 1. the mass spectrogram that contains the histidine peptide that is enriched to from the red proteolytic cleavage peptide section of horse cardiac muscle, band * person is for containing the histidine peptide.
Fig. 2. contain the second order ms figure of histidine peptide (VEADIAGHGQEVLIR, 17-3 1).
Fig. 3. cut the mass spectrogram that contains the histidine peptide that is enriched to the peptide section from cow's milk globulin enzyme, band * person is for containing the histidine peptide.
Fig. 4. contain histidine peptide (ALPMHIR, second order ms figure 142-148).
Embodiment
The following examples will be further explained the present invention, but the present invention is not limited only to these embodiment, the scope that these embodiment do not limit the present invention in any way.Some change that those skilled in the art is made within the scope of the claims and adjust also should be thought and belongs to scope of the present invention.
The enrichment and the order-checking of peptide fragment containing histidine during embodiment 1 horse cardiac muscle red eggs are white
1.1 it is white to get 1mg horse cardiac muscle red eggs, is dissolved in the borate solution of 1mL 0.2M pH 8.Myoglobins does not contain cysteine residues, so can save the reductive alkylation step.Add 20ug trypsase, 37 ℃ of enzymes were cut 12 hours.Adding 0.1M o-methyl-isourea, transfer to 11,37 ℃ of reactions of pH after 4 hours with NaOH, is 8 with the hydrochloric acid adjust pH again, adds the o-methyl benzoic acid anhydride of 20mM, room temperature reaction 1 hour.
1.2 on Yi Lite P230 liquid chromatograph (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.), with C18-reverse-phase chromatographic column (200 * 4.6mm, 5um, 300 , Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.) and strong cation exchange chromatographic column (200 * 4.6mm, 5um, 300 , Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.) series connection, carry out the enrichment of histidine peptide section.Earlier with 0.1% trifluoroacetic acid (TFA) balance chromatographic system, 0.1% trifluoroacetic acid (TFA) with 10 times of column volumes behind the last sample is washed the post desalination, use 60% methyl alcohol (containing 1% acetate) to wash post again, use 60% methyl alcohol (containing 0.5M sodium chloride, 1% acetate) wash-out at last and collect fraction.
1.3 mass spectrophotometry is carried out having equipped on the 4700 Proteomics Analyzer mass spectrometers (u.s.a. applied biosystem company) of nitrogen laser (337nm).Point 0.6uL matrix solution (5mg/mL alpha-cyano-4-hydroxycinnamic acid is with the 50% acetonitrile preparation that contains 0.1%TFA) on the sample target is put the 0.6uL fraction after doing again earlier, carries out mass spectrophotometry after volatilizing naturally.Carry out the external mass calibration with the peptide reference material earlier, the accuracy of mass measurement is generally ± 0.2Da, gathers the one-level mass spectrogram under the negative ion reflective-mode, and visible 1495.7,1602.9,1726.7,1788.9,2109.1 grades contain the [M-H of histidine peptide
+]
-The mass spectra peak (see figure 1).Switch to then under the positive ion reflective-mode, select [the M+H of corresponding peptides
+]
+Ion carries out the tandem mass spectrum analysis as parent ion, can be observed the good second order ms figure of quality, and fragmention is based on the y ion.For example, can be observed all y type ion (see figure 2)s of y1 to y15 in 1790.9 the mass spectrogram.
Contain the enrichment and the order-checking of histidine peptide in the embodiment 2 cow's milk globulin
2.1 get 1mg cow's milk globulin, be dissolved in the borate solution (containing the 6M guanidine hydrochloride) of 0.1mL 0.2M pH 8.Add 10mM dithiothreitol (DTT) (DTT), 37 ℃ of reactions were cooled to room temperature after 1 hour, added 20mM iodoacetamide (IAA), and the reaction of room temperature lucifuge adds 10mM DTT cessation reaction after 1 hour.Behind 6 times of the dilute with waters, add 20ug trypsase, 37 ℃ of enzymes were cut 12 hours.Adding 0.1M o-methyl-isourea, transfer to 11,37 ℃ of reactions of pH after 4 hours with NaOH, is 8 with the hydrochloric acid adjust pH again, adds the o-methyl benzoic acid anhydride of 20mM, room temperature reaction 1 hour.
2.2 on Yi Lite P230 liquid chromatograph (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.), with C18-reverse-phase chromatographic column (200 * 4.6mm, 5um, 300 , Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.) and strong cation exchange chromatographic column (200 * 4.6mm, 5um, 300 , Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.) series connection, carry out the enrichment of histidine peptide section.Earlier with 0.1% trifluoroacetic acid (TFA) balance chromatographic system, 0.1% trifluoroacetic acid (TFA) with 10 times of column volumes behind the last sample is washed the post desalination, use 60% methyl alcohol (containing 1% acetate) to wash post again, use 60% methyl alcohol (containing 0.5M sodium chloride, 1% acetate) wash-out at last and collect fraction.
2.3 mass spectrophotometry is carried out having equipped on the 4700 Proteomics Analyzer mass spectrometers (u.s.a. applied biosystem company) of nitrogen laser (337nm).Point 0.6uL matrix solution (5mg/mL alpha-cyano-4-hydroxycinnamic acid is with the 50% acetonitrile preparation that contains 0.1%TFA) on the sample target is put the 0.6uL fraction after doing again earlier, carries out mass spectrophotometry after volatilizing naturally.Carry out the external mass calibration with the peptide reference material earlier, the accuracy of mass measurement is generally ± 0.2Da, gathers the one-level mass spectrogram under the negative ion reflective-mode, and visible 1019.6 for containing [the M-H of histidine peptide
+]
-The mass spectra peak (see figure 3).Switch to then under the positive ion reflective-mode, select its [M+H
+]
+Ion carries out the tandem mass spectrum analysis as parent ion, can be observed the good second order ms figure of quality, and fragmention is main (see figure 4) with the y ion.
List of references:
1.Ji?J，Chakraborty?A，Geng?M，Zhang?X，Amini?A，Bina?M，Regnier?F.J?Chromatogr?BBiomed?Sci?Appl.2000，745(1)：197-210；
2.Hansen?P，Lindeberg?G.J?Chromatogr?A.1995，690(2)：155-9；
3.Chaga，G.S.J.Biochem.Biophys.Metgods?2001，49，313-334；
4.Amini?A，Chakraborty?A，Regnier?F?E.J?Chromatogr?B?Analyt?Technol?Biomed?Life?Sci.2002，772(1)：35-44
5.Ren?D，Penner?NA，Slentz?BE，Mirzaei?H，Regnier?F.J?Proteome?Res.2003，2(3)：321-9.
6.Keough?T，Youngquist?RS，Lacey?MP.Proc?Natl?Acad?Sci?USA.1999，96，7131-7136.；
7.Keough?T，Lacey?M?P?and?Youngquist?R?S.Rapid?Commun.Mass?Spectrom.2000，14，2348-2356。
Claims (9)
1. method that is used for peptide fragment containing histidine enrichment and order-checking, this method comprises:
(1) opens disulfide bond in the protein molecule with reductive agent, destroy its higher structure; Seal sulfydryl with alkylating reagent, prevent that it from forming disulfide bond again;
(2) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry; Protect with the side chain amino that reagent is cut the peptide section to enzyme;
(3) the peptide section is carried out chemical modification and processing and carried out mass spectrophotometry;
The disposal route that it is characterized in that described step (3) is:
(a) use one or more chemical reagent, the N end amino of peptide section is modified, make it be with acidic-group;
(b) peptide section sample is carried out desalination;
(c) contain the histidine peptide with cation-exchange chromatography post separation and concentration;
(d) make the mass spectrogram that contains histidine peptide cracking generation fragmention with mass spectrum, mass spectrometric data is resolved, identify to produce the protein that contains the histidine peptide.
2. the method for claim 1 is characterized in that acidic-group described in the step (a) is selected from one or more in the following sulfonic acid: 2-sulfo group acetyl group, 3-sulfo group propiono, 2-sulfo group benzoyl or 3-sulfo group benzoyl.
3. the method for claim 1, the method that it is characterized in that desalination described in the step (b) is a reversed phase chromatography.
4. the method for claim 1 is characterized in that mass spectrum described in the step (d) is selected from MALDI tandem mass spectrum, electro-spray ionization tandem mass spectrum or liquid-matter coupling tandem mass spectrum.
5. the method for claim 1 is characterized in that the parsing of mass spectrometric data described in the step (d) and the authentication method of protein are commodity in use software or database search.
6. the method for claim 1 is characterized in that the disposal route of step (3) is:
(a) the sulfonic acylating reagent of apparatus is modified the N end amino of peptide section, makes it be with acidic-group;
(b) with the on-line coupling reversed phase chromatography peptide section sample is carried out desalination;
(c) contain the histidine peptide with cation-exchange chromatography post separation and concentration;
(d) make the mass spectrogram that contains histidine peptide cracking generation fragmention with liquid-matter coupling tandem mass spectrum, commodity in use software or database search are resolved mass spectrometric data, identify to produce the protein that contains the histidine peptide.
7. kit that is used for peptide fragment containing histidine enrichment and order-checking is characterized in that the component of this kit comprises:
(a) one or more N end amino to the peptide section are modified, and make it with the reagent of going up acidic-group;
(b) one or more can carry out the equipment that the desalination of peptide section separates with cation-exchange chromatography;
(c) one or more are used for the buffer system that the desalination of peptide section separates with cation-exchange chromatography.
8. kit as claimed in claim 7 is characterized in that the described reagent of component (a) is the sulfonic acylating reagent of tool.
9. kit as claimed in claim 7 is characterized in that the described equipment of component (b) is that on-line coupling type conventional analysis chromatographic column or rifle hair style are filled pillar.
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