CN106868080A - A kind of method of selective enrichment N- ends serine/threonine peptide fragment - Google Patents
A kind of method of selective enrichment N- ends serine/threonine peptide fragment Download PDFInfo
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Abstract
The present invention relates to a kind of method of N- ends serine/threonine peptide fragment in selective enrichment protein sample enzymolysis liquid based on hydrazide chemistry method.Take biological sample, digested using trypsase Trypsin, and remove sugar chain with PNGase F enzymes, then by the peptide fragment selective oxidation that the N- ends in protein enzymatic hydrolyzate are serine or threonine it is peptide fragment that N- ends are aldehyde radical structure with sodium metaperiodate, processed using the peptide fragment after hydrazide materials enrichment oxidation and using hydroxylamine hydrochloride afterwards, LC-MS/MS analyses are finally carried out to the peptide fragment for discharging to obtain the qualification result of peptide fragment and albumen.Due to only selective enrichment serine/threonine peptide fragment in N- ends therein, therefore can be used for the simplification of protein group sample enzymolysis liquid.The enrichment method principle is simple, easily operation, and enrichment specificity is high.
Description
Technical field
The invention belongs to proteomics research direction complex biological sample preprocess method technical field, specifically
It is related to method and its application of the hydrazide chemistry method for selective enrichment N- ends serine/threonine peptide fragment.
Background technology
" shotgun " is the mainstream technology of current proteomics research, in this technology, complicated albumen
Matter group sample digests into the mixture of peptide with enzyme (mainly trypsase) first, then with one-dimensional or many
Dimension isolation technics is separated to the zymolyte, and is detected with second order mses；Utilize obtained series connection
Mass spectrogram, albumen (document 1.Yates, J.R., The Revolution is identified by database retrieval
and Evolution of Shotgun Proteomics for Large-Scale Proteome Analysis.J Am
ChemSoc, 2013,135 (5), 1629-1640.).But this technology there is also some problems, an albumen warp
Crossing enzymolysis can averagely produce tens kinds of peptide fragments, therefore a protein group sample for complexity by enzymolysis later
The mixture of produced peptide is and its complexity that may contain the different peptide (document of hundreds of thousands kind afterwards
2.Zhang,H.；Li,X.J.；Martin,D.B.；Aebersold,R.,Identification and
quantification of N-linked glycoproteins using hydrazide chemistry,stable
isotope labeling and mass spectrometry.Nature biotechnology,2003,21(6),660-
666.).Bulk redundancy peptide fragment so from high-abundance proteins interferes with the detection and identification of low-abundance protein.
In order to solve this problem, a relatively good strategy is to carry out richness by the feature peptide fragment to protein
Collection simplifies come the enzymolysis liquid to protein, removes the peptide fragment of redundancy, so as to reduce the intensity for separating analysis.
In the world, several samples preprocess method has been developed at present to simplify protein group sample enzymolysis liquid
Complexity, wherein containing specific rare amino acid residue such as cysteine (document 3.Giron, P.；Dayon,
L.；David,F.；Sanchez,J.C.；Rose,K.,Enrichment of N-terminal cysteinyl-
Peptides by covalent capture.J Proteomics, 2009,71 (6), 647-61.), histidine (document
4.Mesmin,C.；Domon,B.,Improvement of the Performance of Targeted LC-MS Assays
through Enrichment of Histidine-Containing Peptides.J Proteome Res,2014,13(12),
6160-6168.), tryptophan (document 5.Yu, Y.；Liu,M.；Yan,G.；He,Y.；Xu,C.；Shen,
H.；Yang,P.,Hydrazide-functionalized magnetic microspheres for the selective
enrichment of digested tryptophan-containing peptides in serum.Talanta,2011,85
(2), 1001-1006.) etc. peptide fragment to be enriched with out be the more commonly used method.And these specific amino
The presence of sour residue can also improve the reliability of Identification of Fusion Protein.Except above-mentioned amino acid residue, N- terminal filament ammonia
Acid/threonine peptide fragment is also more special, the serine at its N- end or 1, the 2- amino alcohols structure of threonine with
Syn diol structure in glycopeptide is similar to, it is also possible to by sodium periodate oxidation be aldehyde radical, so as to by hydrazide materials
Capture.Thus be accordingly used in hydrazide chemistry method (the document 6.Nilsson, J. of enrichment glycopeptide；Ruetschi,U.；
Halim,A.；Hesse,C.；Carlsohn,E.；Brinkmalm,G.；Larson,G.,Enrichment of
glycopeptides for glycan structure and attachment site identification.Nature
Methods, 2009,6 (11), 809-U26.) can be used for the enrichment of N- ends serine/threonine peptide fragment.
The present invention exactly develops a kind of enrichment protein group based on solid-phase capture-release using hydrazide chemistry method
The method of N- ends serine/threonine peptide fragment in sample enzymolysis liquid.By sodium periodate oxidation, hydrazide materials
Capture, hydroxylamine hydrochloride release (document 7.Dirksen, A.；Yegneswaran,S.；Dawson,P.
E.,Bisarylhydrazones as exchangeable biocompatible
Linkers.AngewandteChemie, 2010,49 (11), 2023-7.), finally there was only N- ends serine/threonine
Peptide fragment carries out LC-MS/MS analyses.Substantial amounts of redundancy peptide fragment is so eliminated, so as to greatly reduce sample
Complexity be beneficial to the identification and analysis of low-abundance protein.
The content of the invention
It is an object of the invention to provide it is a kind of can with it is easy, efficiently, the enrichment complexity albumen sample of high specific
The method of N- ends serine/threonine peptide fragment in product protein enzymatic hydrolyzate.
The present invention provide method be using hydrazide materials can with the N- terminal filaments propylhomoserin after sodium periodate oxidation/
Aldehyde radical on threonine peptide fragment reacts, so as to by N- ends serine/threonine peptide fragment from complex sample egg
Concentration and separation out, then obtains the peptide fragment and Identification of Fusion Protein result of sample by mass spectral analysis in white enzymolysis liquid.
The present invention is adopted the following technical scheme that：
Biological sample is taken, is digested using trypsase Trypsin, and sugar chain is removed with PNGase F enzymes,
Then it is by the peptide fragment selective oxidation that the N- ends in protein enzymatic hydrolyzate are serine/threonine with sodium metaperiodate
N- ends are the peptide fragment of aldehyde radical structure, and the peptide fragment after being aoxidized using hydrazide materials enrichment afterwards simultaneously uses hydroxylamine hydrochloride
Treatment, finally carries out LC-MS/MS analyses to obtain the qualification result of peptide fragment and albumen to the peptide fragment for discharging.
Described peptide fragment selective enrichment method is comprised the following steps that：
(1) biological sample is taken, the cushioning liquid of Urea containing 6-8M and 50-100mM TEAB is dissolved in
In, final concentration of 10-20mM DTT, 1-3h in 37-60 DEG C of water-bath are added, it is subsequently adding final concentration
It is 20-40mM IAA, 20-25 DEG C of lucifuge reacts 40-60min, concentration of the biological sample in cushioning liquid
It is 1-10mg/mL；
(2) sample that step (1) is obtained after terminating, being pressed with the aqueous solution containing 50-100mM TEAB will
Urea concentration dilutions are subsequently adding with albumen quality than being 1/50-1/25's to the dilution proportion of 0.5-1M
Trypsase Trypsin, 12-16h is digested in 37 DEG C of water-baths, obtains peptide fragment solution；
(3) sample that step (2) is obtained after terminating, is existed with the super filter tube that molecular cut off is 3-30K
Centrifugal force obtains filter liquor to carry out ultrafiltration centrifugation under 2000-20000g；
(4) it is 500Unites/mg- to be added in the solution for obtaining to step (3) with peptide fragment mass ratio
The PNGase F of 1000Unites/mg, are subsequently placed in digestion 1-10h in 37 DEG C of water-baths；
(5) trifluoroacetic acid is added to adjust pH to 2-3, Ran Houyong in the peptide fragment solution for obtaining to step (4)
C18 solid-phase extraction columns remove the small molecule in solution, the peptide fragment eluent freeze-drying that will be obtained；
(6) the protein group sample peptide hydrolysis for obtaining 100-1000 μ g steps (5) redissolve in
In the oxidation buffer solution of 100-1000 μ L；
(7) to sodium metaperiodate is added in the solution of above-mentioned steps (6), its final concentration of 0.2-20mM is made,
The lucifuge reaction 10-100min at a temperature of 4-37 DEG C；
(8) to sodium thiosulfate is added in the solution of above-mentioned steps (7), its final concentration of 0.4-40 is made
MM, reacts 10-30min at a temperature of 4-37 DEG C；
(9) in the solution of above-mentioned steps (8) being added into 10-200 μ L hydrazide materials, on the oscillator
It is vortexed after shaking 12-36h and supernatant is removed by the method for centrifugation, supernatant is non-N- terminal filaments ammonia
The mixed solution of acid/threonine peptide fragment；
(10) in the hydrazide materials for adding above-mentioned steps (9) to obtain wash solution, whirlpool on the oscillator
Supernatant is removed by the method for centrifugation after rotation concussion 1-10min, supernatant is non-on hydrazide materials
The mixed solution of the peptide fragment of specific adsorption；
(11) 100-1000 μ L hydroxylamine hydrochlorides are added in the hydrazide materials for obtaining to above-mentioned steps (10)
In solution, 12-36h is incubated in 25-40 DEG C of water-bath, then by centrifugation, collects upper strata clear
Liquid, adds trifluoroacetic acid to adjust pH to 2-3, then removes the small molecule in solution with C18 solid-phase extraction columns,
The peptide fragment eluent freeze-drying that will be obtained be enriched with after N- ends serine/threonine peptide fragment.
The solution composition of step (6) oxidation buffer be 10-200mM sodium acetates and 10-200mM sodium chloride,
Remaining is water, and pH value is 3.0-6.5.
Temperature control is at 10-40 DEG C when step (9) mesoscale eddies vibrates；
Step (9), step (10) and in step (11) centrifuge centrifugal force be 500g-3000g.
Wash solution in step (10) is followed successively by 10-300mM sodium-chloride water solutions, 50%-100% acetonitriles
The aqueous solution and 0.1%-3% sodium-chloride water solutions, continuous washing 1-6 times respectively of every kind of wash solution.
The composition of the hydroxylamine hydrochloride solution in step (11) is 10-200mM sodium acetates and 50-300mM salt
Sour azanol, remaining is water, and pH value is 2.5-6.5.
The N- ends serine/threonine peptide fragment that will be obtained in above-mentioned steps (11) is redissolved in 0.1% volumetric concentration
Formic acid in carry out RP LC-MS/MS analyses.
The method specificity is high, can reduce the complexity of enzymolysis liquid, promotes the identification of low-abundance protein, can use
In the Proteomic analysis of complex biological sample.
Described peptide fragment selective enrichment method, peptide fragment enrichment material used is the hydrazides modification of commercialization
Ago-Gel (75-300 μm) (Hercules, CA, U.S.A.).
Described peptide fragment selective enrichment method, only has N- ends serine/threonine peptide fragment in protein enzymatic hydrolyzate
Can just be selectively oxidized, so as to be enriched with by hydrazide materials.
Advantages of the present invention：
The enrichment method is simple, it is easy to operate, and specificity is high, and qualification result has very high before and after example enrichment
Complementarity, is a kind of sequence-specific enrichment method.The present invention is that hydrazide materials are used for into N- terminal filament ammonia first
The enrichment of acid/threonine peptide fragment, with reference to high-resolution RPLC-MS/MS analyses, reduces the complexity of sample
Degree, is conducive to the identification and analysis of low-abundance protein.The characteristic of its sequence-specific enrichment is in analysing protein
Cell component, the aspect such as the molecular action of protein has very big application prospect.
Brief description of the drawings
Fig. 1 is N- ends serine/threonine peptide fragment enrichment method in the complex proteins group sample enzymolysis liquid
Flow chart.The peptide fragment mixture obtained after enzymolysis is aoxidized with sodium metaperiodate, the mixture and acyl for obtaining
Hydrazine material carries out vortex oscillation, after the redundancy peptide fragment for washing away non-specific adsorption, will be with using hydroxylamine hydrochloride
The peptide fragment that microballoon is combined is released and carries out LC-MS/MS analyses.
Fig. 2 is N- ends serine/threonine peptide fragment enrichment method in the complex proteins group sample enzymolysis liquid,
For the peptide fragment appraising datum of mouse liver protein group credit analysis, experimental group (sample after enrichment) and control group
(sample before enrichment) has all carried out three technologies and has repeated to test respectively.The identification of peptide fragment before and after (a) enrichment
As a result.The Weblogo figures of peptide fragment are identified before and after (b) enrichment.
Fig. 3 is N- ends serine/threonine peptide fragment enrichment method in the complex proteins group sample enzymolysis liquid,
For the identification of proteins data of mouse liver protein group credit analysis.A () is enriched with first three albumen of technology repetition
Matter identifies number, three identification of proteins numbers of technology repetition after (b) enrichment, albumen before and after (c) enrichment
The overlap of matter qualification result.
Fig. 4 is N- ends serine/threonine peptide fragment enrichment method in the complex proteins group sample enzymolysis liquid,
For the identification of proteins result of human serum protein group credit analysis.Experimental group (sample after enrichment) and right
Three technologies are all carried out respectively according to group (sample before enrichment) to repeat to test, the result in figure is three skills
Art repeats the average value of experiment.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to institute
Among the scope of embodiments stated.
Embodiment 1
N- ends serine/threonine peptide fragment enrichment method is analysed for the credit of mouse liver protein group：
(1) 1mg mouse liver protein samples are taken, Urea containing 8M and 100mM TEAB (tetrems is dissolved in
Base ammonium bromide) cushioning liquid in (pH 8.0), final concentration of 20mM DTT are added, in 60 DEG C of water-baths
1h, is subsequently adding final concentration of 40mM IAA, and 25 DEG C of lucifuges react 40min to close sulfydryl；
(2) Urea concentration is diluted to 1M with 100mM TEAB (pH 8.0), be subsequently adding 25 μ g
Trypsase, 16h is digested in 37 DEG C of water-baths；
(3) with the super filter tube that molecular cut off is 10K under 14000g centrifugal force to obtained above molten
Liquid carries out ultrafiltration centrifugation, obtains filter liquor；
(4) the PNGase F enzymes of 500Unites are added, digestion 6h in 37 DEG C of water-baths is subsequently placed in；
(5) pH to 2.5 is adjusted with trifluoroacetic acid, then C18 solid-phase extraction columns remove the small molecule in solution,
The peptide fragment eluent freeze-drying that will be obtained；
(6) peptide hydrolysis that 200 μ g are obtained are redissolved in 400 μ L oxidation buffer solution, is added eventually
Concentration is the sodium metaperiodate of 2mM, lucifuge reaction 60min at a temperature of 25 DEG C；It is subsequently adding the thio sulphur of 4mM
Sour sodium, reacts 15min to be quenched oxidation reaction at 25 DEG C；
(7) by above-mentioned solution 40 μ L hydrazide materials of addition, be vortexed concussion 24 on 25 DEG C of oscillators
H, is centrifuged removal supernatant under 1400g centrifugal actions afterwards；
(8) it is each continuous with the 150mM NaCl aqueous solution, 80% (v/v) acetonitrile, the 0.9%NaCl aqueous solution
Wash above-mentioned hydrazide materials 3 times, to wash away the peptide fragment of non-specific adsorption, and hydrazide materials are dispersed in
In the 200 μ L hydroxylamine hydrochloride aqueous solution, 16h is incubated in 37 DEG C of water-baths；
(9) supernatant liquor is collected, pH to 2.5 is adjusted with trifluoroacetic acid, then removed with C18 solid-phase extraction columns
The small molecule gone in solution, the N- terminal filaments propylhomoserin that the peptide fragment eluent freeze-drying that will be obtained is enriched with/
Threonine peptide fragment mixture；
(10) peptide fragment mixture obtained above is redissolved with 0.1% (v/v) formic acid carries out RP LC-
MS/MS is analyzed.
Fig. 2 is the qualification result of peptide fragment, peptide fragment identification number and N- terminal filament propylhomoserins total before and after contrast enrichment
Ratio shared by/threonine peptide fragment, it can be seen that enrichment after peptide fragment sum reduce but N- terminal filaments propylhomoserin/
The ratio of threonine peptide fragment but increases to 93.5% by 15.0% before enrichment, illustrates this solid-phase capture-release
Putting that N- ends serine/threonine peptide fragment is enriched with out by method from complex proteins group sample enzymolysis liquid can be with
The complexity of sample is effectively reduced, and with enrichment specificity very high.Can be more by Weblogo figures
Intuitively find out that the specificity of the method is very high, but also be a kind of sequence-specific enrichment method.
Fig. 3 is the qualification result of protein, and three technologies are repeated before being as can be seen from the figure enriched with and after enrichment
The overlap of qualification result is very high, there is 60%~70%, but before merging enrichment respectively and three after enrichment
The result that secondary technology is repeated, then contrast and find that both overlap only have 37%, so low overlap
There is complementarity very high with the qualification result before enrichment after illustrating enrichment.Also, it is also found that from figure
There is more than 500 albumen newly to be identified after being enriched with, because the abundance of these albumen is relatively low, Direct Analysis
It is not easy arriving for identification.And the enrichment method selectivity N- ends serine/threonine peptide fragment is enriched with out,
High-abundance proteins redundancy peptide fragment is eliminated, so as to be conducive to the identification of low-abundance protein.
Embodiment 2
N- ends serine/threonine peptide fragment enrichment method is analysed for the credit of human serum protein group：
This experiment human serum used is provided by Subsidiary Second Hospital, Dalian Medical Univ. (Dalian, China).
The acquirement and use of sample are completely legal, and meet the relevant regulations of the Ethics Committee of institute.Human albumin
The enrichment experiment of N- ends serine/threonine peptide fragment is operated with the mouse orgotein experiment flow of embodiment 1 in sample.
Fig. 4 is the proteinogram map number distribution map identified before and after N- ends serine/threonine peptide fragment is enriched with,
As can be seen from the figure the spectrogram number that albumen is identified before and after being enriched with has a very large change, after enrichment
Albumen concentrates on low spectrogram identification number field (less than 8), and spectrogram high identifies number (being more than 32)
Protein total also greatly reduces, such as abundance highest albumen in serum, seralbumin, its spectrogram number
From before enrichment 1583 are reduced to 244 after being enriched with, and this is just illustrated by N- ends serine/threonine peptide fragment
Enrichment can effectively reduce the complexity of haemocyanin sample enzymolysis liquid, so as to be conducive to some in serum
The identification of low-abundance protein.
In a word, the present invention is digested based on hydrazide chemistry method for a kind of come selective enrichment complex proteins group sample
N- ends serine/threonine peptide fragment reduces the complexity of sample in liquid, so as to promote low-abundance protein to identify
Method.The method principle is simple, easily operation, and enrichment specificity is high, can effectively reduce protein group sample
The complexity of product enzymolysis liquid, promotes the identification of low-abundance protein.
Claims (8)
1. a kind of method of selective enrichment N- ends serine/threonine peptide fragment, it is characterised in that：
Biological sample is taken, is digested using trypsase Trypsin, and sugar chain is removed with PNGase F enzymes,
Then with sodium metaperiodate by peptide fragment selective oxidation that the N- ends in protein enzymatic hydrolyzate are serine or threonine
For N- ends are the peptide fragment of aldehyde radical structure, the peptide fragment after being aoxidized using hydrazide materials enrichment afterwards simultaneously uses hydrochloric acid hydroxyl
Amine treatment, one kind or two kinds of mixing in the N- terminal filaments propylhomoserin or threonine peptide fragment that obtain.
2. method according to claim 1, it is characterised in that：
Described peptide fragment selective enrichment is comprised the following steps that：
(1) biological sample is taken, the cushioning liquid of Urea containing 6-8M and 50-100mM TEAB is dissolved in
In, final concentration of 10-20mM DTT, 1-3h in 37-60 DEG C of water-bath are added, it is subsequently adding final concentration
It is 20-40mM IAA, 20-25 DEG C of lucifuge reacts 40-60min, concentration of the biological sample in cushioning liquid
It is 1-10mg/mL；
(2) sample that step (1) is obtained after terminating, being pressed with the aqueous solution containing 50-100mM TEAB will
Urea concentration dilutions are subsequently adding with albumen quality than being 1/50-1/25's to the dilution proportion of 0.5-1M
Trypsase Trypsin, 12-16h is digested in 37 DEG C of water-baths, obtains peptide fragment solution；
(3) sample that step (2) is obtained after terminating, is existed with the super filter tube that molecular cut off is 3-30K
Centrifugal force obtains filter liquor to carry out ultrafiltration centrifugation under 2000-20000g；
(4) it is 500Unites/mg- to be added in the solution for obtaining to step (3) with peptide fragment mass ratio
The PNGase F of 1000Unites/mg, are subsequently placed in digestion 1-10h in 37 DEG C of water-baths；
(5) trifluoroacetic acid is added to adjust pH to 2-3, Ran Houyong in the peptide fragment solution for obtaining to step (4)
C18 solid-phase extraction columns remove the small molecule in solution, the peptide fragment eluent freeze-drying that will be obtained；
(6) the protein group sample peptide hydrolysis for obtaining 100-1000 μ g steps (5) redissolve in
In the oxidation buffer solution of 100-1000 μ L；
(7) to sodium metaperiodate is added in the solution of above-mentioned steps (6), its final concentration of 0.2-20mM is made,
The lucifuge reaction 10-100min at a temperature of 4-37 DEG C；
(8) to sodium thiosulfate is added in the solution of above-mentioned steps (7), its final concentration of 0.4-40 is made
MM, reacts 10-30min at a temperature of 4-37 DEG C；
(9) in the solution of above-mentioned steps (8) being added into 10-200 μ L hydrazide materials, on the oscillator
It is vortexed after shaking 12-36h and supernatant is removed by the method for centrifugation, supernatant is non-N- terminal filaments ammonia
The mixed solution of acid/threonine peptide fragment；
(10) in the hydrazide materials for adding above-mentioned steps (9) to obtain wash solution, whirlpool on the oscillator
Supernatant is removed by the method for centrifugation after rotation concussion 1-10min, supernatant is non-on hydrazide materials
The mixed solution of the peptide fragment of specific adsorption；
(11) 100-1000 μ L hydroxylamine hydrochlorides are added in the hydrazide materials for obtaining to above-mentioned steps (10)
In solution, 12-36h is incubated in 25-40 DEG C of water-bath, then by centrifugation, collects supernatant liquor,
Add trifluoroacetic acid to adjust pH to 2-3, then remove the small molecule in solution with C18 solid-phase extraction columns, will
To peptide fragment eluent freeze-drying be one kind in N- terminal filaments propylhomoserin or threonine peptide fragment after being enriched with or
Two kinds of mixing.
3. method according to claim 2, it is characterised in that：
The solution composition of step (6) oxidation buffer be 10-200mM sodium acetates and 10-200mM sodium chloride,
Remaining is water, and pH value is 3.0-6.5.
4. method according to claim 2, it is characterised in that：
Temperature control is at 10-40 DEG C when step (9) mesoscale eddies vibrates；
Step (9), step (10) and in step (11) centrifuge centrifugal force be 500g-3000g.
5. method according to claim 2, it is characterised in that：
Wash solution in step (10) is followed successively by 10-300mM sodium-chloride water solutions, 50%-100% second
The nitrile aqueous solution and 0.1%-3% sodium-chloride water solutions, continuous washing 1-6 times respectively of every kind of wash solution.
6. method according to claim 2, it is characterised in that：Hydroxylamine hydrochloride in step (11)
The composition of solution is 10-200mM sodium acetates and 50-300mM hydroxylamine hydrochlorides, and remaining is water, and pH value is
2.5-6.5。
7. method according to claim 1 and 2, it is characterised in that：
The N- ends serine/threonine peptide fragment that will be obtained redissolves and RP is carried out in the formic acid of 0.1% volumetric concentration
LC-MS/MS is analyzed.
8. method according to claim 1 and 2, it is characterised in that：The method specificity is high, only
Having N- ends serine/threonine peptide fragment can be selectively oxidized, so as to be enriched with by hydrazide materials, therefore energy
The complexity of enzymolysis liquid is reduced, promotes the identification of low-abundance protein, can be used for the protein of complex biological sample
Group analysis.
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| CN113358873A (en) * | 2020-03-04 | 2021-09-07 | 中国科学院大连化学物理研究所 | Method for improving mass spectrum detection sensitivity of D-dimer in sample based on ultrafiltration-assisted enzymolysis |
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| CN1516741A (en) * | 2001-04-03 | 2004-07-28 | ����Ī�����ɷ�����˾ | Methods and kits useful for simplification of complex peptide mixtures |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1516741A (en) * | 2001-04-03 | 2004-07-28 | ����Ī�����ɷ�����˾ | Methods and kits useful for simplification of complex peptide mixtures |
Non-Patent Citations (1)
| Title |
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| YATING YAO等: "Specific Enrichment of Peptides with N-Terminal Serine/Threonine by a Solid-Phase Capture-Release Approach for Efficient Proteomics Analysis", 《ANALYTICAL CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113358873A (en) * | 2020-03-04 | 2021-09-07 | 中国科学院大连化学物理研究所 | Method for improving mass spectrum detection sensitivity of D-dimer in sample based on ultrafiltration-assisted enzymolysis |
| CN113358873B (en) * | 2020-03-04 | 2022-09-27 | 中国科学院大连化学物理研究所 | Method for improving mass spectrum detection sensitivity of D-dimer in sample based on ultrafiltration-assisted enzymolysis |
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