CN1314968C - Trace polypeptide or protein-enriched and its direct analyzing method - Google Patents
Trace polypeptide or protein-enriched and its direct analyzing method Download PDFInfo
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- CN1314968C CN1314968C CNB2004100168899A CN200410016889A CN1314968C CN 1314968 C CN1314968 C CN 1314968C CN B2004100168899 A CNB2004100168899 A CN B2004100168899A CN 200410016889 A CN200410016889 A CN 200410016889A CN 1314968 C CN1314968 C CN 1314968C
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Abstract
The present invention relates to a method for enrichment of trace amount peptide and protein samples and direct analysis of matrix assisting and laser analyzing ionization mass spectra by using zeolite nanometer particles as micro nanometer adsorbing agents. The zeolite nanometer particles have good adsorption effect on protein and polypeptide, and have good compatibility with the matrix assisting and laser analyzing ionization mass spectra; the process of matrix assisting and laser analyzing ionization mass spectrographic analysis can be directly carried out for samples which are adsorbed by the zeolite nanometer particles without a sample eluting step; thus, the operation is simple, and sample loss caused by the sample eluting step is avoided. The enriching efficiency of the present invention for polypeptide can reach more than 1250 times farthest, and a protein enzymolysis sample of 50*10<-12> g/uL can also be identified by the method which is provided by the present invention.
Description
Technical field
The invention belongs to biochemical analysis field, is the method for a kind of trace samplings enrichment and evaluation.Utilize a series of zeolite nano particles, polypeptide and protein sample are carried out enrichment, and the sample of institute's enrichment is directly carried out the analysis and the evaluation of substance assistant laser desorpted ionization massspectrum with different surfaces character (different crystal structure and silica alumina ratio).
Background technology
The analysis of low-abundance protein with identify it is one of the emphasis of proteomics research and difficult point content.The albumen of bearing important vital movement in biosome often all is low-abundance protein, yet its extremely low content brings difficulty for follow-up analysis and detection, limited research and the understanding of people to them, therefore, realize that effectively concentrating of low-abundance protein is to realize one of its accurate most important condition of analyzing and identifying.In fact all relating to effective enrichment of sample in the proteomics research process aspect many, is example with the analysis of enzymolysis sample in the glue, because the volume of peptide hydrolysis extract is too big, must concentrate before mass spectrophotometry.The most frequently used sample concentration method has solvent evaporated method and chromatogram concentration method at present.Solvent evaporated method mainly contains boulton process and freeze-drying, and these class methods waste time and energy, and vessel surface absorption can cause a large amount of peptide section losses in the dry run, influences the sensitivity that mass spectrum is identified.It is to utilize absorption peptide section such as hydrophilic/hydrophobic interaction that chromatogram concentrates rule, uses a small amount of organic solvent wash-out again, and its operating process is loaded down with trivial details, and the sample that hydrophobicity is strong is difficult to by wash-out, and the hydrophilic peptide section owing to the reverse phase filler acting force a little less than reclaim rate variance.
Summary of the invention
The objective of the invention is to obtain a kind of simple to operate, efficient is high, effective, the new method that can carry out mass spectrophotometry to trace polypeptide or albumen.
A kind of trace polypeptide that the present invention proposes or protein enrichment and direct analytical approach thereof, be to utilize of the absorption of zeolite nano-particle molecular to polypeptide and protein sample, after trace polypeptide or albumen carried out enrichment, need not elution step and directly finish the analyzing and testing of substance assistant laser desorpted ionized flight time mass spectrum, detailed process is:
(1) with the zeolite nano particle as nano adsorber, add the aqueous solution enrichment of polypeptide or protein example, sample concentration is 2 * 10
-14-2 * 10
-10G/ μ L, enrichment time is between 60-180 minute, and the enrichment temperature is between 20-37 degree centigrade, and zeolite nano particle amount is a 10-1000 μ g/1mL sample;
(2) be need not wash-out by the sample of enrichment, directly evenly mix, form the crystallization of uniform and delicate, carry out substance assistant laser desorpted ionized flying time mass spectrum analysis with matrix;
(3), identify polypeptide or protein according to the mass spectrum result.
Process of the present invention is as follows:
1 selects the zeolite nano particle of dissimilar and silica alumina ratio as the sample adsorbent.
2 utilize selected zeolite nano particle to carry out the enrichment of trace amount of protein or polypeptide.
3 need not wash-out, directly carry out MALDI-TOF/MS and analyze.
Actual conditions is as follows:
Temperature was bathed 60-180 minute to impel sample fully to adsorb under the extremely dilute solution of 1 a certain amount of zeolite nano particle and protein or peptide was blended in 20-37 ℃.
2 centrifugal back reject supernatants are collected lower floor's zeolite facies, dilute with suitable quantity of water again.
Zeolite nano particle after the 3 an amount of enriched sample and organic substrate for example 2-cyano group-4-hydroxycinnamic acid (after α-CHCA) mixes, carry out substance assistant laser desorpted ionized flight time mass spectrum and measure.
The particle diameter of zeolite nano particle of the present invention is that the 10-200nm scope is comparatively suitable.Such zeolite particles not only has the performance of big crystal grain zeolite, also has bigger characteristics such as active outside surface, the ion-exchange performance that enriches adjustable surface nature, outside surface uniqueness and the absorption of equally distributed aperture.
The present invention utilizes the surface nature of zeolite nano particle, reach the purpose of enrichment by acting force and biomolecule action between Van der Waals force, hydrogen bond, Electrostatic Absorption, hydrophilic/hydrophobic effect equimolecular, synergy by multiple acting force, overcome the sample absorption discrimination effect that single absorption mode causes, has good universality, by certain acting force of conciliation or modification of zeolite particle, this has better targeted.The surface nature difference of zeolite nano particle of the present invention is different with accumulation ability to the absorption of polypeptide or albumen, therefore can be according to same zeolite nano particle of the autotelic selection of the character for the treatment of enriched sample or multiple zeolite nano particle mixing enrichment, multiple nano particle mixes can be by reconciling or modify wherein a kind of acting force of reinforcement, make its tool with certain specificity, for example can be by increasing the content of zeolite crystal aluminium, improve the negative charge density of zeolite surface, and then improve ability to function the high sample of isoelectric point.
The present invention's silica alumina ratio is that 60 β (β-60) or silicalite-1 zeolite nano particle are best as adsorbent.Enrichment time of the present invention was at 60-180 minute, and the enrichment temperature is at 25-35 ℃, and sample concentration is 2 * 10
-14-2 * 10
-10G/ μ L, the addition of zeolite nano particle is a 10-1000 μ g/1mL sample.The present invention can form the adjustable stable colloidal solution of concentration with any zeolite nano particle, forms uniform and stable enrichment solution with albumen and polypeptide solution the low bulky living things system of content is had better suitable tonality, helps the carrying out of enrichment.
The present invention evenly mixes the careful crystallization of generation with zeolite nano particle and organic substrate, being formed with of the mixed crystal of zeolite and matrix is beneficial to matrix the laser energy that is absorbed is transferred to by the sample of zeolite adsorption, realizes that the ground substance assistant laser of sample is resolved ionization process; The crystalline solid of uniform and delicate has guaranteed the reappearance of sample analysis and result's reliability simultaneously.
The present invention can it all has inrichment to polypeptide or albumen with any zeolite nano particle, can be uniformly mixed to form the crystallization of uniform and delicate with matrix, and it is little that substance assistant laser desorpted ionization/flight time mass spectrum is measured influence.This method is simple to operate, efficient is high, effective.
Description of drawings
Fig. 1 is the sem photograph of zeolite nano particle β-60.
Fig. 2 is the sem photograph of zeolite nano particle silicalite-1.
Fig. 3 and 0.25 μ L 50pg/ μ L mark peptide mixer and the mixed crystallization figure of isopyknic α-CHCA saturated solution on the MALDI target plate.
Fig. 4 is equal-volume (0.25 μ L) 50pg/ μ L mark peptide mixer, 10mg/mL β-60 zeolite nano particle and the α-mixed crystallization figure of CHCA saturated solution potpourri on the MALDI target plate.
Fig. 5 is equal-volume (0.25 μ L) 50pg/ μ L mark peptide mixer, 10mg/mL β-60 zeolite nano particle and the α-crystallization figure of CHCA saturated solution potpourri on the MALDI target plate.Comparison diagram 3 and Fig. 4,5 zeolite nano particle as can be seen can generate even, careful crystallization with organic substrate.
Fig. 6 is the MALDI-TOF/MS spectrogram of 50pg/ μ L mark peptide mixer.
Fig. 7 is the MALDI-TOF/MS spectrogram under the silicalite-1 zeolite nano particle mixing point batten spare of 50pg/ μ L mark peptide mixer and equal-volume 10mg/mL.Comparison diagram 6 and Fig. 7 as can be seen, the MALDI-TOF/MS that the adding of nano zeolite particle does not influence sample measures
Fig. 8 is the MALDI-TOF/MS spectrogram of zeolite nano particle β-60 enrichment standard polypeptide sample.Sample concentration, 40fg/ μ L, experimental technique is according to example 8.
Fig. 9 is the MALDI-TOF/MS spectrogram of zeolite nano particle β-60 enrichment standard polypeptide sample.Sample concentration, 40fg/ μ L, experimental technique is according to example 9.The zeolite nano particle can be above 1250 times to the thickening efficiency of peptide 1 as can be seen from Fig. 8,9 results.
Figure 10 is the MALDI-TOF/MS spectrogram of the white enzymolysis sample of zeolite nano particle enrichment horse cardiac muscle red eggs.Sample concentration, 50pg/ μ L, experimental technique is according to example 22.
Figure 11 is the MALDI-TOF/MS spectrogram of the white enzymolysis sample of zeolite nano particle enrichment horse cardiac muscle red eggs.Sample concentration, 50pg/ μ L, experimental technique is according to example 22.
Embodiment
Following example is that zeolite nanometer particle material provided by the present invention is carried out further specifying of example enrichment and the direct analytic process of substance assistant laser desorpted ionized flight time mass spectrum.
The influence that example 1-5 zeolite nano particle is measured the substance assistant laser desorpted ionization-flight time mass spectrum (MALDI-TOF/MS) of mark peptide mixer
Get three kinds of marks of 50pg/ μ L peptide mixed solution, 0.25 μ L and put respectively to the MALDI target plate for five parts, add 0.25 μ L H more successively
2The suspending liquid (20mg/mL) of O, LTL, β-30, β-60 and silicalite-1, the last saturated solution (0.1%TFA, 50% acetonitrile solution) that covers isopyknic α-CHCA successively after the drying to be crystallized, carries out mass spectrophotometry.Used mass spectrum MALDI-TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems); Laser instrument is a Nd-YAG laser, wavelength 355nm, laser pulse frequency 200Hz; Accelerating potential 20kV; Positive ion mode, reflective TOF detects.
Example 6-9 polypeptide sample enrichment and mass spectroscopy
Get the 20mg/mL zeolite nano particle LTL that 1ml polypeptide sample aqueous solution adds 5 μ L respectively, β-30, the suspension suspending liquid of β-60 and silicalite-1 is at 37 ℃ of 90min that vibrate down; Centrifugal 10min under the 13000g, the reject supernatant adds 5 μ L H in the zeolite precipitation
2O, vibration makes it to suspend.The saturated solution mixing point of suspending liquid 0.25 μ L and equal-volume α-CHCA carries out mass spectroscopy according to example 1-5 condition to the MALDI target plate.
Example 10-14
The addition of adjusting the zeolite nano particle of 20mg/mL is 1,2,10,20,50 μ L, and other conditions are constant, repeat above-mentioned concentrating and the mass spectrum experiment.
Example 15-17
Adjusting adsorption time is 30,60,120min, and other conditions concentrate-the mass spectrum experiment with example 6-9.
Example 18-19
Adjusting adsorption temp is 20,45 ℃, and other conditions concentrate-the mass spectrum experiment with example 6-9.
The evaluation of example 20-23 protein digestion thing
Protein is got the NH of 5mg/ml horse cardiac muscle red eggs white (myoglobin) according to the method enzymolysis of document
4HCO
3Solution (20mmol/L), heating is 5 minutes in the boiling water, to impel protein denaturation, the room temperature cooling.Add tryptic solution with 50: 1 ratio of substrate/enzyme mass ratio, 37 ℃ of following isothermal reactions are more than 12 hours.Product is diluted to 50pg/ μ L, gets the LTL that the 0.5mL sample adds the 20mg/mL of 5 μ L respectively, β-30, the suspension of β-60 and silicalite-1 carries out example enrichment according to the method for example 6-9, carries out mass spectrophotometry according to the method for example 20.The mass spectrum result carries out database retrieval (http: ∥ prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) by proteinprospectors.
Following table is the character of three kinds of standard polypeptide using in the example
| The standard polypeptide | Title | Amino acid sequence | Isoelectric point | Molecular weight |
| Peptide 1 | KHHDKHDH | 8.62 | 955.07 | |
| Peptide 2 | Arg 8-Vasotocin | CYQNCPRG | 8.06 | 1050.22 |
| Peptide 3 | Angiotensin I | DRVYIHPFHL | 6.92 | 1296.49 |
Subscript is the white sample data library searching of the horse cardiac muscle red eggs result of example 20-23
| The zeolite nano particle | Qualification result | Sequence coverage rate (%) | Coupling peptide hop count order | |
| The Swiss-port numbering | Title | |||
| LIL | P02128 | MYG_HORSE | 16 | 2 |
| β-30 | P02128 | MYG_HORSE | 28 | 4 |
| β-60 | P02128 | MYG_HORSE | 56 | 9 |
| silicalite-1 | P02128 | MYG_HORSE | 70 | 10 |
Claims (5)
1. trace polypeptide or protein enrichment and directly analytical approach, it is characterized in that utilizing of the absorption of zeolite nano-particle molecular to polypeptide and protein sample, after trace polypeptide or albumen carried out enrichment, need not elution step and directly finish the analyzing and testing of substance assistant laser desorpted ionized flight time mass spectrum, detailed process is:
(1) with the zeolite nano particle as nano adsorber, add the aqueous solution enrichment of polypeptide or protein example, sample concentration is 2 * 10
-14-2 * 10
-10G/ μ L, enrichment time is between 60-180 minute, and the enrichment temperature is between 20-37 degree centigrade, and zeolite nano particle amount is a 10-1000 μ g/1mL sample;
(2) be need not wash-out by the sample of enrichment, directly evenly mix, form the crystallization of uniform and delicate, carry out substance assistant laser desorpted ionized flying time mass spectrum analysis with matrix;
(3), identify polypeptide or protein according to the mass spectrum result.
2. trace polypeptide according to claim 1 or protein enrichment and direct analytical approach thereof, the particle diameter that it is characterized in that the zeolite nano particle is 10-200nm.
3. trace polypeptide according to claim 1 or protein enrichment and direct analytical approach thereof is characterized in that the temperature that the zeolite nano particle carries out enriched sample is 25-37 ℃.
4. trace polypeptide according to claim 1 or protein enrichment and direct analytical approach thereof is characterized in that the zeolite nano particle is β-60 or silicalite-1.
5. trace polypeptide according to claim 1 or protein enrichment and direct analytical approach thereof is characterized in that selecting the potpourri of one or more zeolite nano particles to carry out the enrichment of trace polypeptide or protein.
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Families Citing this family (14)
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| CN1333250C (en) * | 2005-10-20 | 2007-08-22 | 复旦大学 | Proteolytic method for desorpting ionization source mass-spectrum target plate based on matrix auxiliary laser |
| CN100374857C (en) * | 2006-01-26 | 2008-03-12 | 复旦大学 | Method for fast enriching trace polypeptide and protein and realizing identification |
| CN101368890B (en) * | 2007-08-17 | 2011-06-22 | 复旦大学 | Method for in-situ desalination and enrichment on trace amount of protein or polypeptide target |
| CN101464430B (en) * | 2007-12-21 | 2011-11-23 | 中国科学院大连化学物理研究所 | Method and special apparatus for on-line enrichment and automatic analysis of endogenous polypeptide |
| CN101220128B (en) * | 2008-01-31 | 2011-04-06 | 复旦大学 | Polymethyl methacrylate composite microsphere, preparation method and application thereof |
| CN101943688B (en) * | 2009-07-10 | 2013-01-02 | 复旦大学 | Method for enriching glycosylated peptide by utilizing mass spectrum target plate |
| CN102072932B (en) * | 2009-11-19 | 2012-12-12 | 复旦大学 | Method and device for identifying glycopeptide segment |
| CN103884574B (en) * | 2012-12-19 | 2016-06-29 | 中国科学院大连化学物理研究所 | A kind of integrated protein C-end enrichment method |
| CN103454371B (en) * | 2013-07-23 | 2015-10-07 | 复旦大学 | Leaf proteins based on one dimension long column Liquid Chromatography-Tandem Mass Spectrometry is separated authentication method |
| CN103743847A (en) * | 2013-12-31 | 2014-04-23 | 张金玲 | MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) detection method of fluoroquinolone medicines in milk |
| CN107356699B (en) * | 2017-07-18 | 2019-08-09 | 浙江省肿瘤医院 | In rich plasma or serum in 12 kinds abundance and low-abundance protein method |
| CN107202850B (en) * | 2017-07-18 | 2019-08-09 | 浙江省肿瘤医院 | The method of 5 kinds of low-abundance proteins in rich plasma or serum |
| DE102019126201A1 (en) * | 2019-09-27 | 2021-04-01 | Bruker Daltonik Gmbh | Mass spectrometric sample preparation for matrix-assisted ionization (e.g. MALDI) |
| CN113687003B (en) * | 2021-09-14 | 2022-06-14 | 谱天（天津）生物科技有限公司 | Method for improving identification number of protein and/or peptide fragment group mass spectrum |
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