CN101363779A - Method for enhancing ionization efficiency of peptide segment - Google Patents
Method for enhancing ionization efficiency of peptide segment Download PDFInfo
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- CN101363779A CN101363779A CNA2008100396982A CN200810039698A CN101363779A CN 101363779 A CN101363779 A CN 101363779A CN A2008100396982 A CNA2008100396982 A CN A2008100396982A CN 200810039698 A CN200810039698 A CN 200810039698A CN 101363779 A CN101363779 A CN 101363779A
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Abstract
The invention belongs to the biochemical analysis field and relates to a method for improving the peptide segment ionization efficiency. An organic compound with alkalescence and hydrophobic nature is utilized to conduct derivation for protein or peptide segment carboxyl, so as to improve the peptide segment ionization efficiency. All derived samples perform mass spectrometric analysis and identification directly. The organic compound selected in the invention can form a covalent bond with the peptide segment carboxyl with high efficiency and high specificity, thereby effectively improving the combining capacity of the peptide segment and protons, greatly improving the peptide segment ionization efficiency, and enabling the peptide segment with low abundance ratio and difficult ionization to be detected. The invention has the advantages of high reaction specialty, few secondary reaction, simple steps, economy, time-saving, and the like. The invention can be widely used in the proteomics field, and greatly improve the detection capacity of mass spectrum for the peptide segment with low abundance ratio and difficult ionization.
Description
Technical field
The invention belongs to biochemical analysis field, relate to a kind of method that improves ionization efficiency of peptide segment.Be specifically related to utilize alkalescence and hydrophobic organic compound that the carboxyl of polypeptide and protein sample is derived, make its mass spectrum Ionization Efficiency be improved, and the sample of being derived is directly carried out mass spectrophotometry and evaluation.
Background technology
Be accompanied by the enforcement and the propelling of the Human Genome Project (HGP), life science has entered the genome times afterwards comprehensively.Present stage, the main research object of life science is a functional genomics, comprises research of structural gene group and proteome research etc.Illustrate the expression rule and the biological function of all protein of the expressed real execution vital movement of genome, compare with the rule of illustrating the gene activity on the genome integral level, task is more complicated, and is also arduous more.
Known in this field, proteomics research comprises the Separation of Proteins in sample or the tissue and identifies two committed steps.Along with new soft ionization mode---the generation of ground substance assistant laser desorption ionization (MALDI) and electron spray ionisation (ESI), biological mass spectrometry has become one of of paramount importance technological means in the proteomic techniques platform.Protein group is an integral body of in time and space dynamic change, and the sample system is extremely complicated, and albumen DYNAMIC DISTRIBUTION scope is very wide.The protein relevant with the signal conduction with disease often belongs to low-abundance protein, these important protein matter are owing to the amount that itself exists seldom is difficult to be able to effective evaluation, and the posttranslational modification that extensively exists in the sample particularly phosphorylation modification is low and be subjected to the reasons such as compacting of non-phosphorylating peptide section easily owing to itself Ionization Efficiency, make that the direct evaluation for phosphated peptide section and protein exists very big difficulty.
Along with the further expansion of proteome research and deeply, especially the Analysis and Identification of difficult ionised peptide section such as phosphorylation modification and low abundance proteins to the mass spectrum ionization techniques more and more higher requirement is proposed.Existing method at difficult ionization phosphated peptide section mainly is to carry out specific enrichment for phosphated peptide section, such as adopting IMAC, TiO
2Select and concentrated phosphoric acid peptide section Deng enrichment material, thereby reach compacting of removing non-phosphorylating peptide section and the purpose that improves concentration.Yet the specificity of these methods is also not high enough, and complex steps is consuming time also long, has increased sample loss, contamination of heavy greatly, and has not been suitable for high-throughout identification of proteins.And for low abundance proteins, enrichment remains topmost method, and this method needs a large amount of samples to do support, and the also very difficult removal of the compacting of high-abundance proteins matter, causes its Mass Spectrometer Method is not still well improved at present.
Summary of the invention
The object of the present invention is to provide a kind of method that fundamentally improves peptide section mass spectrum Ionization Efficiency.Be specifically related to a kind of deriving and improve the method for ionization efficiency of peptide segment by peptide section carboxyl.This method is simple to operate, highly sensitive, favorable reproducibility, economy, save time, be adapted to high throughput protein group research requirement, can directly carry out mass spectrophotometry to phosphated peptide section or low abundance proteins.
For achieving the above object, the present invention adopts following technical scheme:
1, utilize alkalescence and hydrophobic organic compound, to polypeptide or protein example (be called for short: the carboxyl peptide section) is derived, and realizes improving polypeptide mass spectrum Ionization Efficiency, it is characterized in that by following method and step:
1) be dissolved in alkalescence and hydrophobic organic compound, peptide bond condensation reagent and catalyzer in the organic solvent respectively, described three kinds of reagent add successively in order in the described peptide section aqueous solution and form reaction system, with acid for adjusting pH value 7.5～7.8, above-mentioned reaction system is put suspendible under the room temperature;
2) the described solvent in the above-mentioned reaction system of removal stops derivatization reaction, gets derivative products;
3) with above-mentioned derivative products dissolving, be directly used in MALDI-MS or ESI-MS and detect.
The carboxyl of polypeptide of the present invention and protein example comprises one of carbon tip carboxyl and acidic amino acid side chain carboxyl group.
Alkalescence of the present invention and hydrophobic organic compound are the compounds that contains pyrimidine radicals, are selected from 2-diazanyl pyrimidine, 1-(2-pyrimidine radicals) piperazine, 4-amino-2-(1-piperazinyl) pyrimidine or 4-amino-6-chlorine pyrimidine.
Peptide bond condensation reagent of the present invention is selected from N, N-dicyclohexylcarbodiimide, N, N-diisopropyl carbon, diimine 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride or phosphinylidyne diimidazole.
Catalyzer of the present invention is selected from I-hydroxybenzotriazole, 6-chloro-I-hydroxybenzotriazole or 1-hydroxyl-7-azepine benzotriazole.
Acid such as trifluoroacetic acid, hydrochloric acid, formic acid or acetate are adopted in acid of the present invention.
The present invention adopts means such as natural drying at room temperature, oven dry, vacuum drying or nitrogen dry up to remove reaction dissolvent by removing the purpose that solvent in the reaction system reaches cessation reaction.
The present invention directly is used for the electric spray ion source Mass Spectrometer Method with derivative products with the aqueous solution dissolving that contains 5% acetonitrile, 0.1% formic acid, or derivative products is used for substance assistant laser desorpted ion source massspectrum detection with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid.
Thereby the inventive method has effectively changed the character of peptide section by electronegative, hydrophilic carboxyl in alkalescence and the hydrophobic organic compound change peptide section, and its ionizing efficiency is improved, and has improved its Mass Spectrometer Method sensitivity greatly.For example for phosphated peptide section (TPpTAPSLG), the back Ionization Efficiency has improved 101 times through deriving.
The compounds that contains pyrimidine radicals that the present invention selects for use is as the organic compound of the carboxyl of deriving, because pyrimidine radicals gas phase basicity height, hydrophobicity be strong, at the ultraviolet end absorption is arranged, so in case be applied to the MALDI ionization, it can effectively promote the interaction of peptide section and matrix, the auxiliary laser that absorbs; And for the ESI ionization, high gas phase basicity also can effectively be improved the ionizing efficiency of peptide section.Therefore, adopt the compound contain pyrimidine radicals can effectively realize improving the effect of the Ionization Efficiency of peptide section as the organic compound of the carboxyl of deriving.
Description of drawings
Fig. 1 is the MALDI-TOF-MS spectrogram after peptide section KRGSGAW process is derived,
Wherein, demonstrate reaction efficiency nearly 100%.
Fig. 2 is the MALDI-TOF-MS spectrogram of peptide section RPPGFSP and TPpTAPSLG,
Wherein, the Ionization Efficiency of peptide section RPPGFSP has improved 12.1 times, and the Ionization Efficiency of peptide section TPpTAPSLG has improved 101 times.
Fig. 3 is the multiple that the Ionization Efficiency of seven standard peptide sections improves,
Wherein, shown that the Ionization Efficiency of non-phosphorylating peptide section has improved 10 to 50 times, the Ionization Efficiency of phosphated peptide section has improved 50 to 101 times.
Fig. 4 is the mixed MALDI-TOF-MS spectrogram of the bovine serum albumin(BSA) of standard phosphoeptide and trypsin digestion, and wherein (a) do not pass through the spectrogram of deriving, and (b) is the spectrogram through deriving; The peptide section of the bovine serum albumin(BSA) that is not identified before asterisk is illustrated in and derives; The peptide section of the bovine serum albumin(BSA) that is all identified before and after the numbering expression; Demonstrate, through deriving, the signal intensity of all peptide sections all gets a promotion, and the amplitude maximum that improves of phosphated peptide section wherein is difficult to detect before deriving, and becomes a strongest peptide section of intensity after deriving.
Fig. 5 is that the peptide section increased substantially figure as a result through the back Ionization Efficiency of deriving during ESI-MS detected,
Wherein, 5a is peptide section TPpTALSLG, and 5b is peptide section YMQSpTPL1, and 5c is peptide section QYGSGAW.
Embodiment
Following example is that utilization alkalescence and hydrophobic organic compound that the present invention proposes are derived to the carboxyl of polypeptide and protein sample, make its mass spectrum Ionization Efficiency be improved, and the sample of being derived is directly carried out further specifying of mass spectrophotometry and evaluation.
The reaction efficiency of embodiment 1 checking derivatization reaction is near 100%
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section KRGSGAW aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Above-mentioned dried product exhibited is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows that derivatization reaction efficient is about 100% (Fig. 1).
The reaction efficiency of embodiment 2 checking derivatization reactions is near 100%
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DRVYIHPF aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Above-mentioned dried product exhibited is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, draw two site derivatization reaction efficient near 100%.
The multiple that embodiment 3 peptide section RPPGFSP improve through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section RPPGFSP aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows that 12.1 times (Fig. 2 is a) in the Ionization Efficiency raising of peptide section.
The multiple that embodiment 4 peptide section TPpTAPSLG improve through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section TPpTAPSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows, 101 times (Fig. 2 b) of Ionization Efficiency raising of peptide section.
The multiple that embodiment 5 peptide section KRGSGAW improve through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section KRGSGAW aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows, 40 times (Fig. 3) of Ionization Efficiency raising of peptide section.
The multiple that embodiment 6 peptide section NRGSGAW improve through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section NRGSGAW aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows, 15 times (Fig. 3) of Ionization Efficiency raising of this peptide section.
The multiple that embodiment 7 peptide section DPpTALSLG improve through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows, 99 times (Fig. 3) of Ionization Efficiency raising of this peptide section.
The multiple that embodiment 8 peptide section TPpTAPpSLG improve through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid detects, the result shows, 47 times (Fig. 3) of Ionization Efficiency raising of this peptide section.
1-(2-pyrimidine radicals) piperazine is diluted in N, and in the dinethylformamide, with N, N-dicyclohexylcarbodiimide and 1-hydroxyl-7-azo benzotriazole is dissolved in N respectively, in the dinethylformamide, is mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 94 times, N is described, the N-dicyclohexylcarbodiimide can reach effect same as the peptide bond condensation reagent.
Embodiment 10 peptide section TPpTAPSLG adopt N, the multiple that Ionization Efficiency improved after N-diisopropyl carbon was derived as the peptide bond condensation reagent
1-(2-pyrimidine radicals) piperazine is diluted in N, and in the dinethylformamide, with N, N-diisopropyl carbon and 1-hydroxyl-7-azo benzotriazole is dissolved in N respectively, in the dinethylformamide, is mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 90 times, N is described, N-diisopropyl carbon can reach effect same as the peptide bond condensation reagent.
The multiple that Ionization Efficiency improved after embodiment 11 peptide section TPpTAPSLG adopted the phosphinylidyne diimidazole to derive as the peptide bond condensation reagent
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, phosphinylidyne diimidazole and 1-hydroxyl-7-azo benzotriazole is dissolved in N respectively, in the dinethylformamide, be mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 85 times, illustrates that the phosphinylidyne diimidazole can reach effect same as the peptide bond condensation reagent.
The multiple that Ionization Efficiency improved after embodiment 12 peptide section TPpTAPSLG adopted 6-chloro-I-hydroxybenzotriazole to derive as catalyzer
1-(2-pyrimidine radicals) piperazine is diluted in N, and in the dinethylformamide, with N, N-dicyclohexylcarbodiimide and 6-chloro-I-hydroxybenzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 87 times, illustrates with 6-chloro-I-hydroxybenzotriazole can reach same effect as catalyzer.
The multiple that Ionization Efficiency improved after embodiment 13 peptide section TPpTAPSLG adopted 1-hydroxyl-7-azepine benzotriazole to derive as catalyzer
1-(2-pyrimidine radicals) piperazine is diluted in N, and in the dinethylformamide, with N, N-dicyclohexylcarbodiimide and 1-hydroxyl-7-azepine benzotriazole is dissolved in N respectively, in the dinethylformamide, is mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 93 times, illustrates with 1-hydroxyl-7-azepine benzotriazole can reach same effect as catalyzer.
The multiple that Ionization Efficiency improved after embodiment 14 peptide section TPpTAPSLG adopted 2-diazanyl pyrimidine to derive as derivative reagent
2-diazanyl pyrimidine is released in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azepine benzotriazole are dissolved in N respectively, in the dinethylformamide, be mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 96 times, illustrates that 2-diazanyl pyrimidine can be used as derivative reagent.
The multiple that Ionization Efficiency improved after embodiment 15 peptide section TPpTAPSLG adopted 4-amino-2-(1-piperazinyl) pyrimidine to derive as derivative reagent
4-amino-2-(1-piperazinyl) pyrimidine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azepine benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section DPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of this peptide section improves 90 times, illustrates that 4-amino-2-(1-piperazinyl) pyrimidine can be used as derivative reagent.
Embodiment 16 peptide section TPpTAPSLG adopt formic acid to regulate the multiple that pH improves through the back Ionization Efficiency of deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section TPpTAPSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the aqueous formic acid with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of peptide section improves 101 times, illustrates that formic acid can be used for regulating pH.
Embodiment 17 peptide section TPpTAPSLG adopt nitrogen to dry up, the multiple that Ionization Efficiency improves through after deriving
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section TPpTAPSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.Nitrogen dries up then, removes all solvents.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for MALDI-MS with the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be detected, the Ionization Efficiency of peptide section improves 101 times, illustrates that nitrogen dries up to can be used for cessation reaction.
Under the embodiment 18 checking mixed systems, derive to the influence of enzymolysis protein and phosphated peptide section
Adjusting sample solution is the standard phosphated peptide section mixed solution (solvent is 50% acetonitrile, 50% water, 0.1% trifluoroacetic acid) of 15nmol bovine serum albumin(BSA) peptide hydrolysis and 2pmol, repeat derivatization reaction and mass spectrum experiment in the example 2, the result shows, phosphoeptide can't detect substantially before derivatization reaction, phosphoeptide signal intensity the strongest (Fig. 4) behind the derivatization reaction.
Embodiment 19 checkings peptide section TPpTALSLG in the electron spray ionisation source increases substantially through the back Ionization Efficiency of deriving.
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section TPpTALSLG aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.Adding in the peptide section derivative products of each is waited the not derived peptide segment of amount of substance, above-mentioned product is used for ESI-MS with the aqueous solution dissolving that contains 5% acetonitrile, 0.1% formic acid detects, the result shows, the Ionization Efficiency of this peptide section increases substantially that (Fig. 5 a).
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section YMQSpTPL aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for ESI-MS with the aqueous solution dissolving that contains 5% acetonitrile, 0.1% formic acid detects, the result shows that the Ionization Efficiency of this peptide section increases substantially (Fig. 5 b).
Embodiment 21 checkings peptide section QYGSGAW in the electron spray ionisation source increases substantially through the back Ionization Efficiency of deriving.
1-(2-pyrimidine radicals) piperazine is diluted in N, in the dinethylformamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 1-hydroxyl-7-azo benzotriazole are dissolved in N respectively, in the dinethylformamide, are mixed with 2 milligrams every milliliter solution.Above three kinds of reagent are added 6,4 and 3 microlitres successively be about in the peptide section QYGSGAW aqueous solution of the every microlitre of 100 nanograms in 50 microlitre concentration, the trifluoroacetic acid aqueous solution with 0.1% is regulated between the pH value to 7.5 and 7.8.With above-mentioned mixed system suspendible 30 seconds at room temperature.The traditional vacuum drying is removed all solvents then.With the not derived peptide segment of amount of substances such as adding in the peptide section derivative products of each, above-mentioned product is used for ESI-MS with the aqueous solution dissolving that contains 5% acetonitrile, 0.1% formic acid detects, the result shows that the Ionization Efficiency of this peptide section increases substantially (Fig. 5 c).
Claims (9)
1. method that improves ionization efficiency of peptide segment is characterized in that by following method and step:
1) utilize alkalescence and hydrophobic organic compound, the carboxyl of polypeptide or protein derived,
Adopt alkalescence and hydrophobic organic compound, peptide bond condensation reagent and catalyzer to be dissolved in the organic solvent respectively, above-mentioned three kinds of reagent add successively in polypeptide or the protein aqueous solution in order and form reaction system, with acid for adjusting pH value to 7.5～7.8, suspendible under the room temperature;
2) solvent in the above-mentioned reaction system of removal stops derivatization reaction, gets product
3) with above-mentioned product dissolving, be directly used in MALDI-MS or ESI-MS and detect.
2. by the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that the carboxyl of described polypeptide or protein comprises one of carbon tip carboxyl and acidic amino acid side chain carboxyl group.
3. press the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that described alkalescence and hydrophobic organic compound are the compounds that contains pyrimidine radicals, be selected from 2-diazanyl pyrimidine, 1-(2-pyrimidine radicals) piperazine, 4-amino-2-(1-piperazinyl) pyrimidine or 4-amino-6-chlorine pyrimidine.
4. by the method for the described raising ionization efficiency of peptide segment of claim 3, it is characterized in that the described compound that contains pyrimidine radicals is 1-(2-pyrimidine radicals) piperazine.
5. press the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that described peptide bond condensation reagent is selected from N, N-dicyclohexylcarbodiimide, N, N-diisopropyl carbon, diimine 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride or and phosphinylidyne diimidazole.
6. by the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that described catalyzer is selected from I-hydroxybenzotriazole, 6-chloro-I-hydroxybenzotriazole or 1-hydroxyl-7-azepine benzotriazole.
7. by the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that described acid is selected from trifluoroacetic acid, hydrochloric acid, formic acid or acetate.
8. by the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that described step 2) the removal reaction system in the mode of solvent be that natural drying at room temperature, oven dry, vacuum drying or nitrogen dry up mode.
9. press the method for the described raising ionization efficiency of peptide segment of claim 1, it is characterized in that directly product being used for the electric spray ion source Mass Spectrometer Method with the aqueous solution dissolving that contains 5% acetonitrile, 0.1% formic acid in the described step 3), or directly product is used the aqueous solution dissolving that contains 50% acetonitrile, 0.1% trifluoroacetic acid to be used for substance assistant laser desorpted ion source massspectrum and detected.
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