CN101042376A - Method and reagent kit for rapid measuring protein N terminal sequence - Google Patents
Method and reagent kit for rapid measuring protein N terminal sequence Download PDFInfo
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- CN101042376A CN101042376A CN 200610065078 CN200610065078A CN101042376A CN 101042376 A CN101042376 A CN 101042376A CN 200610065078 CN200610065078 CN 200610065078 CN 200610065078 A CN200610065078 A CN 200610065078A CN 101042376 A CN101042376 A CN 101042376A
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Abstract
This invention provides one new method and agent case for rapid protein N end sequence testing, which comprises the following: protein amidogen decorating, restoring alkyl base and pancreatic enzymes cutting, enzyme peptides sections mass spectrum analysis, mass spectrum identification and test sequence, wherein, through the selective decoration for easy determining N end peptides sections and for easy testing sequence for gene engineer product N end sequence. This invention also describes this method agent case.
Description
Technical field
The present invention relates to a kind of sequence measurement of protein.Specifically, the measuring method of mass spectrum that relates to protein N terminal sequence.The invention still further relates to the kit that is used for this method.
Background technology
As everyone knows, the order-checking of the N of protein end is all extremely important to the evaluation and the functional analysis of protein.On the one hand, the N terminal sequence label of protein has very high specificity.According to statistics, 43% to 83% protein has unique N and holds 4 residue labels (different according to species) [Wilkins, M.R., Gasteiger, E., Tonella, L., Ou, K.et al, J.Mol.Biol.1998,278,599-608.].On the other hand, the N terminal sequence of protein helps to prove conclusively the N end processing of protein, as the removal of signal peptide, the excision of N end methionine residue etc.At present, the method for the most frequently used mensuration protein N terminal sequence is the Edman edman degradation Edman.Though also can realize robotization, it is very high to the purity requirement of sample, and is very consuming time, and the flux of method is also very low.
The invention provides a kind of protein N terminal order-checking simply fast, both can carried out, again can be simultaneously other peptide section be analyzed high throughput method with identification of protein.
Summary of the invention
The invention provides a kind of simple effectively, be used for the new method and the kit of fast measuring protein N terminal sequence.The inventor finds, by introducing electronegative sulfonic group at the N of protein end, both can determine N end peptide section in the negative ion mode mass spectrum, can carry out the mass spectrum order-checking to the peptide section in the positive ion mode mass spectrum again.
Specifically, this method relates to the mass spectrophotometry that modification, reductive alkylation and pancreatin enzyme are cut, enzyme is cut the peptide section of protein amino, the mass spectrum identification and order-checking of N end peptide section, and step comprises:
(1) the side chain amino of protein is modified, with the selectivity of raising modification reaction and the length of the N end peptide section that enzyme is cut generation;
(2) amino with the N of one or more reagent derived proteins end, to introduce pKa less than 2 acidic-group.
(3) open 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;
(4) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry;
(5) determine protein N terminal peptide section with mass-spectrometric technique, the Xingqi sequential analysis of going forward side by side.
The disposal route that it is characterized in that described step (2) is:
(a) with sulfonic acid the terminal amino group of protein is carried out chemical modification, make it be with acidic-group;
(b) contain the chemical reagent of free amine group with one or more, the modification reaction of protein is stopped.
Wherein, the described acidic-group of step (a) is selected from 2-sulfo group acetyl group, 3-sulfo group propiono, 2-sulfo group benzoyl or 3-sulfo group benzoyl.
This method is by introducing acid stronger group at the N of protein end, makes its ionization and detected especially easily in the negative ion mode mass spectrum, need not separate and determines fast N end peptide section to realize the evaluation and the order-checking of N end of protein then by mass-spectrometric technique.Be added in that acidic-group on the protein N terminal peptide section loses proton especially easily and electronegative, thereby in negative ion mode, be very easy to ionization and, be easy to distinguish mutually with other peptide section of protein by Mass Spectrometer Method.In mass spectrophotometry, the acidic-group of introducing can promote the cracking of peptide section ion, simplifies its second order ms figure, thereby helps its sequential analysis.
Utilize this method, the speed and the flux level of protein N terminal order-checking have been improved greatly, significantly increased the lysis efficiency of N end peptide in second order ms, 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 carry out de novo sequencing to protein terminal peptide according to second order ms figure.
The present invention also provides a kind of kit that is used for the fast measuring protein N terminal sequence, and this kit contains:
(a) one or more can be modified protein N terminal amino, make it with the reagent of going up acidic-group;
(b) one or more contain the chemical reagent of free amine group;
Wherein the described reagent of component (a) preferably has sulfonic acylating reagent; Preferred trihydroxyethyl aminomethane of the described chemical reagent of component (b) or lysine.
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 the operation instruction of state administrative organs's approval etc. in addition.
New method of the present invention and kit are applicable to the evaluation of protein and the fast measuring of N terminal sequence; be applicable to the evaluation and the order-checking of N end of protein in biology, medical science, materia medica research and the application; be particularly suitable for the analysis of N terminal sequence and the quality control of genetic engineering recombinant product or medicine, combine with the two dimensional electrophoresis technology and also be applicable to the N end order-checking of protein being carried out scale.
Method of the present invention and kit have practicality widely.Purposes includes but not limited to: the protein 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 negative ion mode peptide quality fingerprinting figure (base peak is a N end peptide section) of back myoglobins is modified in sulfonation
Fig. 2. the tandem mass spectrum figure of back myoglobins N end peptide is modified in sulfonation
Fig. 3. the negative ion mode peptide quality fingerprinting figure that back two kinds of source human growth hormone recombinants are modified in sulfonation compares (mark * person is a N end peptide section)
Fig. 4. the tandem mass spectrum figure that back two kinds of source human growth hormone recombinants' N end peptide is modified in sulfonation compares
Fig. 5. the negative ion mode peptide quality fingerprinting figure (base peak is a N end peptide section) of back lysozyme of chicken is modified in sulfonation
Fig. 6. the tandem mass spectrum figure of back lysozyme of chicken N end peptide is modified in sulfonation
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 N end order-checking that embodiment 1 horse cardiac muscle red eggs are white
1.1 it is white to get 0.1mg horse cardiac muscle red eggs, is dissolved in the borate solution of 0.1mL 0.2M pH 7, adds the o-methyl benzoic acid anhydride of 20mM, room temperature reaction added the Tris/HCl cessation reaction of 100mM pH 8 after 1 hour.Myoglobins does not contain cysteine residues, so can save the reductive alkylation step.Add 2ug trypsase, 37 ℃ of enzymes were cut 8 hours.
1.2 mass spectrophotometry is carried out having equipped on the 4700 Proteomics Analyzer mass spectrometers (u.s.a. applied biosystem company) of nitrogen laser (337nm).After sample and matrix solution (5mg/mL alpha-cyano-4-hydroxycinnamic acid is with the 50% acetonitrile preparation that contains 0.1%TFA) mixed in equal amounts, get the 0.6uL point on the sample target, carry 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 the mass-to-charge ratio of visible base peak is 1998.0, corresponding to [the M-H that modifies back N end peptide
+]
-The quasi-molecular ions (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 y3 to y16 in 1999.9 the mass spectrogram.
The human growth hormone recombinant's of embodiment 2 separate sources N end order-checking is compared with sequence
2.1 get two kinds of each 0.1mg of human growth hormone recombinant's sample of separate sources, be dissolved in the borate solution of 0.1mL 0.2M pH 7 respectively, each adds the o-methyl benzoic acid anhydride of 20mM, and room temperature reaction added the Tris/HCl cessation reaction of 100mM pH 8 after 1 hour.Add 10mM dithiothreitol (DTT) (DTT) again, 37 ℃ of reactions add 2ug trypsase after 1 hour, and 37 ℃ of enzymes were cut 8 hours.
2.2 mass spectrophotometry is carried out having equipped on the 4700 Proteomics Analyzer mass spectrometers (u.s.a. applied biosystem company) of nitrogen laser (337nm).After sample and matrix solution (5mg/mL alpha-cyano-4-hydroxycinnamic acid is with the 50% acetonitrile preparation that contains 0.1%TFA) mixed in equal amounts, get the 0.6uL point on the sample target, carry 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, gather the one-level mass spectrogram under the negative ion reflective-mode, the mass-to-charge ratio of base peak is respectively 1243.5 and 1112.5 in visible two samples, corresponding to [the M-H that modifies back N end peptide separately
+]
-Quasi-molecular ions (seeing band * tagger among Fig. 3 a and the 3b).Switch 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.The mass spectrogram of two samples is compared, be very easy to find the difference of its N terminal sequence: a methionine residue that still keeps the N end, another then is removed (see figure 4).
The N end order-checking of embodiment 3 lysozyme of chicken
3.1 get the 0.1mg lysozyme of chicken, be dissolved in the borate solution (containing 0.1%SDS) of 0.1mL 0.2M pH 7, add the O-methyl-isourea solution of 0.1mL 1M pH 12,37 ℃ were reacted 2 hours.After transferring pH to 7, add the o-methyl benzoic acid anhydride of 20mM, room temperature reaction 1 hour, the Tris/HCl cessation reaction of adding 100mM pH 8.Add 10mM dithiothreitol (DTT) (DTT) again, 37 ℃ of reactions add 2ug trypsase after 1 hour, and 37 ℃ of enzymes were cut 8 hours.
3.2 mass spectrophotometry is carried out having equipped on the 4700 Proteomics Analyzer mass spectrometers (u.s.a. applied biosystem company) of nitrogen laser (337nm).After sample and matrix solution (5mg/mL alpha-cyano-4-hydroxycinnamic acid is with the 50% acetonitrile preparation that contains 0.1%TFA) mixed in equal amounts, get the 0.6uL point on the sample target, carry 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 the mass-to-charge ratio of visible base peak is 830.4, corresponding to [the M-H that modifies back N end peptide
+]
-The quasi-molecular ions (see figure 5).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 6)s of y1 to y5 in 832.4 the mass spectrogram.
Claims (6)
1. method that is used for the fast measuring protein N terminal sequence, this method comprises:
(1) amino to protein carries out chemical modification
(2) open 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;
(3) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry;
(4) with analytical technique of mass spectrum analysing protein terminal peptide fragment, make its cracking produce the mass spectrogram of fragmention;
The disposal route that it is characterized in that described step (1) is:
(a) with sulfonic acid the terminal amino group of protein is carried out chemical modification, make it be with acidic-group;
(b) contain the chemical reagent of free amine group with one or more, the modification reaction of protein is stopped.
2. the method for claim 1 is characterized in that the described acidic-group of step (a) is selected from 2-sulfo group acetyl group, 3 sulfo group propionos, 2-sulfo group benzoyl or 3-sulfo group benzoyl.
3. the method for claim 1 is characterized in that the described chemical reagent of step (b) is selected from trihydroxyethyl aminomethane or lysine.
4. kit that is used for the fast measuring protein N terminal sequence is characterized in that this kit contains:
(a) one or more can be modified protein terminal amino, make it with the reagent of going up acidic-group;
(b) one or more contain the chemical reagent of free amine group;
5. kit as claimed in claim 4 is characterized in that (a) described reagent is for having sulfonic acylating reagent.
6. kit as claimed in claim 4 is characterized in that (b) described chemical reagent is selected from trihydroxyethyl aminomethane or lysine.
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| CN 200610065078 CN101042376A (en) | 2006-03-20 | 2006-03-20 | Method and reagent kit for rapid measuring protein N terminal sequence |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107870215A (en) * | 2017-11-28 | 2018-04-03 | 广东省测试分析研究所（中国广州分析测试中心） | The gas chromatography combined with mass spectrometry analysis method of potassium cinnamate content in a kind of measure food |
| WO2023240563A1 (en) * | 2022-06-16 | 2023-12-21 | 深圳华大智造科技股份有限公司 | Sequencing kit containing thiol blocking reagent, and use of sequencing kit |
-
2006
- 2006-03-20 CN CN 200610065078 patent/CN101042376A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107870215A (en) * | 2017-11-28 | 2018-04-03 | 广东省测试分析研究所（中国广州分析测试中心） | The gas chromatography combined with mass spectrometry analysis method of potassium cinnamate content in a kind of measure food |
| WO2023240563A1 (en) * | 2022-06-16 | 2023-12-21 | 深圳华大智造科技股份有限公司 | Sequencing kit containing thiol blocking reagent, and use of sequencing kit |
| WO2023241701A1 (en) * | 2022-06-16 | 2023-12-21 | 深圳华大智造科技股份有限公司 | Sequencing kit containing sulfydryl blocking reagent and application of sequencing kit |
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