CN103454371A - Proteome separation and identification method based on one-dimensional long column liquid chromatogram tandem mass spectrum - Google Patents
Proteome separation and identification method based on one-dimensional long column liquid chromatogram tandem mass spectrum Download PDFInfo
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Abstract
The invention belongs to the technical field of biology and relates to a proteome separation and identification method based on one-dimensional long column liquid chromatogram tandem mass spectrum. The identification method uses a 50cm reversed phase chromatographic column and adopts a one-dimensional long gradient to separate proteomics samples; one-dimensional proteome separation and identification are carried out by the tandem mass spectrum through electrospray ionization; a use result shows that the identification method is simple in experiment operation and does not need to presort fractions; the identification effect of a traditional two-dimensional mass spectrum can be realized by one time of experiment; particularly, with regard to the treatment of few samples, one time of data analysis of proteomics can be finished by only several micrograms of samples in one time of the experiment; the needed sample amount is few and the instrumental analysis efficiency of complicated biological proteomes can be up to that the analysis and identification of more than 4000 proteins can be realized within 7 hours, so that the rapid and efficient identification of proteins is realized.
Description
Technical field
The invention belongs to biological technical field, relate to the isolation and identification method of protein group, be specifically related to rapidly and efficiently protein component based on one dimension long column Liquid Chromatography-Tandem Mass Spectrometry from authentication method.
Background technology
It is the science of all proteins of a genomic expression of research that prior art discloses proteomics, at first phase is proposed by Marc Wilkins in the early 1990s the earliest, it is the broad scale research to protein, comprises the evaluation of protein, protein post-translational modification and interaction thereof etc.Known, the biological sample of proteomics forms complicated, the complexity that needed to be separated to reduce sample before detecting, method commonly used has the methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing electrophoresis (IEF), reinforcing yin essence/cation exchange (SAX/SCX), reversed-phase liquid chromatography at present.Increase the coverage of protein in order to improve identification rate, researched and proposed a series of two dimensional separation methods; In research practice, often adopt the method for coupling, SDS-PAGE and RPLC, IEF and RPLC, SAX/SCX and RPLC etc. are arranged, can improve the identification number of protein by the coupling between different separation methods, but the separation method length consuming time of two dimension, the consumption sample size is large, complex operation;
In addition, the evaluation of protein is the basis of Study on Protein group, and detecting device commonly used is mass spectrum, and the mass spectrum ionization apparatus comprises the ionization source of substance assistant laser desorpted ionized (MALDI) and electron spray ionisation (ESI) usually; Wherein, the ESI ionization source is because having the ionization apparatus in the compatible Chang Zuowei liquid chromatography mass coupling good with liquid chromatography.
In view of the identification research needs of current protein, the present inventor intends providing a kind of one dimension to separate the proteomics method of identifying.
Summary of the invention
The object of the invention is to overcome the shortcomings and deficiencies of prior art, a kind of isolation and identification method of new protein group is provided, be specifically related to a kind of protein component based on one dimension long column Liquid Chromatography-Tandem Mass Spectrometry from authentication method.The method is the evaluation to the peptide section in conjunction with the one dimension reversed phase chromatography separation advantage of long column and tandem mass spectrum, and the separation that can carry out rapidly and efficiently protein group sample is identified.
For achieving the above object, the present invention adopts following technical scheme:
(1) select suitable protein example preprocess method;
(2) select suitable chromatogram column length;
(3) design suitable chromatographic resolution time and gradient.
Concrete, the protein component based on one dimension long column Liquid Chromatography-Tandem Mass Spectrometry of the present invention, from authentication method, utilizes long chromatographic column to identify 4000 above protein in suitable gradient in 7 hours, and it comprises:
(1) lysate extracts all protein potpourri from cell or tissue;
(2) protein is through precipitation, resuspended, carries out reductive alkylation;
(3) adopt proteinase that the protein mixture enzymolysis of evaluation to be separated is become to peptide section potpourri;
(4) peptide section potpourri enzymolysis obtained carries out the evaluation of one dimension Liquid Chromatography-Tandem Mass Spectrometry.
In authentication method of the present invention, described chromatogram column length is that the chromatographic resolution column length is greater than 40cm, and typical length is 50cm; Described chromatogram gradient is three step-length gradient elutions;
In authentication method of the present invention, in described step (1), lysate comprises 100mM TrisHCl pH 7.6,2% above SDS and DTT and protease inhibitors, and lysate is hatched 5 minutes at high temperature more than 80 degrees centigrade; In one embodiment of the present of invention, preferred described lysate comprises 100mM TrisHCl pH 7.6 and comprises 4%SDS and 100mM DTT and protease inhibitors;
In authentication method of the present invention, in step (2), adopt methyl alcohol-chloroform to be precipitated;
In authentication method of the present invention, be not limited to a kind of enzymolysis of enzyme in step (3), use one or more proteinase combination in any in the enzyme-specifics such as trypsase, GluC or Lys-c to carry out enzymolysis to protein mixture; In one embodiment of the present of invention, preferred, adopt Lys-C and Trypsin combination enzymolysis;
In authentication method of the present invention, in step (4), adopt the 50cm reverse-phase chromatographic column to be identified; Wherein,
Reversed phase chromatography separation column packing particle diameter is 1.8-5 μ m, and the filler porosity is 10nm-50 nm, and the reversed phase chromatography separation column length is greater than 40cm, and typical length is 50cm, and flow velocity is 100-500 nL/min;
In authentication method of the present invention, described three step-length gradient elutions, optimal condition is the 2% acetonitrile 0.1%FA aqueous solution that mobile phase A is pH 2-5, the 98% acetonitrile 0.1%FA aqueous solution that Mobile phase B is pH 2-5, adopted respectively 0-10%B at 0-30 minute; Adopted respectively the gradient of 10-20%B at 30-240 minute; Adopted respectively the gradient of 20-35%B at 240-340 minute; Within 340-345 minute afterwards, all adopt 35-80%B tonsure, 345-355 minute all to adopt the isocratic elution of 80%B, the gradient that 355-360 minute all adopts 80-0%B; Within 360-400 minute, adopt 0%B to carry out balance to chromatographic column.
More specifically, the rapidly and efficiently protein component based on one dimension long column Liquid Chromatography-Tandem Mass Spectrometry of the present invention from authentication method, is characterized in that, comprises step:
(1) cell rinses with PBS, after clean nutrient culture media, uses trypsin digestion cell;
(2) add lysate (100mM TrisHCl pH 7.6 comprises 4%SDS and 100mM DTT and protease inhibitors) to extract, lysate 95 degrees centigrade hatch 5min after, being cooled to the of short duration ultrasonic 20s---of room temperature surpasses 3 seconds and stops 5 seconds, power 100w, 16100g is centrifugal, and 10min gets supernatant, and protein solution is quantitative;
(3) protein obtained in step (1) is used methyl alcohol-chloroform precipitation (to add the long-pending methyl alcohol suspendible of tetraploid, the chloroform suspendible that adds a volume, the deionized water suspendible that adds three volumes, the centrifugal 1min of 14000g, abandon water, add the long-pending methyl alcohol suspendible of limbs, the centrifugal 1min of 14000g, abandon supernatant), precipitation is resuspended in 100mM TrisHCl/6M urea pH 8.5;
(4) protein in 10mM DTT 56 degrees centigrade the reaction 30 minutes, in 20mM IAA, 37 degrees centigrade are reacted 30 minutes, LysC is diluted to urea concentration to the following Trypsin of 2M 1:100 enzymolysis four hours in mass ratio with 50mM ammonium bicarbonate after according to mass ratio 1:50 enzymolysis 3h, adds trypsin and spends the night to the 1:50 enzymolysis; After enzymolysis, add TFA to final concentration 0.1%, stop enzymolysis, desalination, freeze-drying;
(5) peptide section potpourri enzymolysis obtained carries out the evaluation of one dimension Liquid Chromatography-Tandem Mass Spectrometry; The 2% acetonitrile 0.1%FA aqueous solution that the mobile phase A that described quick reversed phase chromatography separation adopts is pH 2-5, the 98% acetonitrile 0.1%FA aqueous solution that Mobile phase B is pH 2-5; Reversed phase chromatography separation column packing particle diameter is 1.8-5 μ m, and the filler porosity is 10nm-50 nm, and the reversed phase chromatography separation column length is greater than 40cm.Flow velocity is 100-500 nL/min; According to the gradient setting, by resulting zymolyte in above-mentioned steps (4), adopted respectively 0-10%B at 0-30 minute; Adopted respectively the gradient of 10-20%B at 30-240 minute; Adopted respectively the gradient of 20-35%B at 240-340 minute; Within 340-345 minute afterwards, all adopt 35-80%B tonsure, 345-355 minute all to adopt the isocratic elution of 80%B, the gradient that 355-360 minute all adopts 80-0%B; Within 360-400 minute, adopt 0%B to carry out balance to chromatographic column.Result is as shown in table 1:
table 1 sample gradient arranges table
| Time/min | %A | %B |
| 0 | 100 | 0 |
| 30 | 90 | 10 |
| 240 | 80 | 20 |
| 340 | 65 | 35 |
| 345 | 20 | 80 |
| 355 | 20 | 80 |
| 360 | 100 | 0 |
| 400 | 100 | 0 |
Compared with prior art, its advantage is authentication method of the present invention:
(1) authentication method of the present invention, the reverse-phase chromatographic column of use 50cm, adopt the long gradient of one dimension to be separated the proteomics sample, and through electron spray ionisation, tandem mass spectrum is identified;
(2) described authentication method experimental implementation is simple, does not need prefrationation to divide, and once experiment can reach the effect that conventional two-dimensional is separated Mass Spectrometric Identification; Particularly, for the processing of a small amount of sample, once experiment only needs a few microgram samples just can complete the data analysis of a proteomics, and required sample size is few.
Authentication method of the present invention be a kind of step simple, easy to operate, efficiently fast the one dimension protein component from authentication method; This authentication method is the evaluation to the peptide section in conjunction with the one dimension reversed phase chromatography separation advantage of long column and tandem mass spectrum, and the separation that protein group sample is carried out is rapidly and efficiently identified; Especially, use the reverse-phase chromatographic column of 50cm, adopt the long gradient of one dimension to be separated the proteomics sample, through electron spray ionisation, tandem mass spectrum is identified, the use result shows, described authentication method experimental implementation simply, does not need prefrationation to divide, and once experiment can reach the effect that conventional two-dimensional is separated Mass Spectrometric Identification; Particularly for the processing of a small amount of sample, once experiment only needs a few microgram samples just can complete the data analysis of a proteomics, required sample size is few, instrumental analysis efficiency to complex biological protein group reaches the Analysis and Identification of realization to 4000 above protein in 7 hours, realizes the rapidly and efficiently evaluation of protein.
The accompanying drawing explanation
The Technology Roadmap that Fig. 1 is the inventive method.
The gradient of the sample that Fig. 2 is embodiment 1 acquisition arranges schematic diagram and total ions chromatogram.
The gradient of the sample that Fig. 3 is embodiment 2 acquisitions arranges schematic diagram and total ions chromatogram.
Embodiment
embodiment 1
The HepG2 cell is by this laboratory cultures; The HepG2 cell is cleaned and to be suspended in the lysate that 100mM TrisHCl pH 7.6 comprises SDS and DTT and protease inhibitors, 95 degrees centigrade hatch 5min after, be cooled to room temperature, of short duration ultrasonic, the centrifuging and taking supernatant, methyl alcohol chloroform precipitation, used Lys-C and trypsase to carry out enzymolysis after the heavy molten albumen of 100mM TrisHCl/6M urea, obtains its peptide section potpourri.
By after 100 μ g albumen mixture enzymolysis of HepG2 cell extraction, get 2 μ g hybrid peptide sections and carry out one dimension reversed phase chromatography separation-esi-msn data acquisition and analysis; The aqueous solution that the mobile phase A that low pH reversed phase chromatography separation adopts is 0.1% formic acid 2% acetonitrile, the aqueous solution that Mobile phase B is 0.1% formic acid 98% acetonitrile; Reversed phase chromatography separation column packing particle diameter is 2 μ m, and filling out footpath is 10nm, and the reversed phase chromatography separation post is 75 μ m * 500 mm.Flow velocity is 200 nL/min.Be arranged on 0-30 minute according to gradient and adopt respectively 0-10%B; Adopted respectively the gradient of 10-20%B at 30-240 minute; Adopted respectively the gradient of 20-35%B at 240-340 minute.Within 340-345 minute afterwards, all adopt 35-80%B tonsure, 345-355 minute all to adopt the isocratic elution of 80%B, the gradient that 355-360 minute all adopts 80-0%B; Within 360-400 minute, adopt 0%B to carry out balance to chromatographic column.The mass spectrum acquisition system is 5600 Q-TOF mass spectrometers (U.S. Applied Biosystems companies), scanning adopts the data dependence pattern: carry out a full scan (sweep limit m/z 350-1500), select 50 the strongest parent ions to carry out secondary scanning (signal threshold value is 150 cps); Electron spray voltage 2300 V, positive ion mode; Obtain total ions chromatogram (as shown in Figure 2); Through the 400min collection analysis, result shows, identifies altogether 4317 protein of 48161 peptide sections.
embodiment 2
HUVEC holoprotein lysate is provided by C-HPP.Methyl alcohol chloroform precipitation, used Lys-C and trypsase to carry out enzymolysis after the heavy molten albumen of 100mM TrisHCl/6M urea, obtains its peptide section potpourri.
By after 100 μ g albumen mixture enzymolysis of HUVEC holoprotein extract, get 4 μ g hybrid peptide sections and carry out one dimension reversed phase chromatography separation-esi-msn data acquisition and analysis; The aqueous solution that the mobile phase A that low pH reversed phase chromatography separation adopts is 0.1% formic acid 2% acetonitrile, the aqueous solution that Mobile phase B is 0.1% formic acid 98% acetonitrile; Reversed phase chromatography separation column packing particle diameter is 2 μ m, and filling out footpath is 10nm, and the reversed phase chromatography separation post is 75 μ m * 500 mm; Flow velocity is 200 nL/min.Be arranged on 0-30 minute according to gradient and adopt respectively 0-10%B; Adopted respectively the gradient of 10-20%B at 30-240 minute; Adopted respectively the gradient of 20-35%B at 240-340 minute; Within 340-345 minute afterwards, all adopt 35-80%B tonsure, 345-355 minute all to adopt the isocratic elution of 80%B, the gradient that 355-360 minute all adopts 80-0%B; Within 360-400 minute, adopt 0%B to carry out balance to chromatographic column; The mass spectrum acquisition system is 5600 Q-TOF mass spectrometers (U.S. Applied Biosystems companies), scanning adopts the data dependence pattern: carry out a full scan (sweep limit m/z 350-1500), select 50 the strongest parent ions to carry out secondary scanning (signal threshold value is 150 cps).Electron spray voltage 2300 V, positive ion mode; The total ions chromatogram obtained (as shown in Figure 3); Through the 400min collection analysis, result shows, identifies altogether 4229 protein of 40735 peptide sections.
Claims (9)
1. the protein component based on one dimension long column Liquid Chromatography-Tandem Mass Spectrometry, from authentication method, is characterized in that, comprises step:
(1) lysate extracts all protein potpourri from cell or tissue;
(2) protein is through precipitation, resuspended, carries out reductive alkylation;
(3) adopt proteinase that the protein mixture enzymolysis of evaluation to be separated is become to peptide section potpourri;
(4) peptide section potpourri enzymolysis obtained carries out the evaluation of one dimension Liquid Chromatography-Tandem Mass Spectrometry;
In described Liquid Chromatography-Tandem Mass Spectrometry, chromatogram column length is that the chromatographic resolution column length is greater than 40cm, and the chromatogram gradient is three step-length gradient elutions.
2. by method claimed in claim 1, it is characterized in that, described chromatographic resolution column length is 50cm.
3. by method claimed in claim 1, it is characterized in that, in described step (1), lysate comprises 100mM TrisHCl pH 7.6,2% above SDS and DTT and protease inhibitors, and lysate is hatched 5 minutes at high temperature more than 80 degrees centigrade.
4. by method claimed in claim 1, it is characterized in that, in described step (1), lysate comprises 100mM TrisHCl pH 7.6 and comprises 4%SDS and 100mM DTT and protease inhibitors.
5. by method claimed in claim 1, it is characterized in that, in described step (2), adopt methyl alcohol-chloroform to be precipitated.
6. by method claimed in claim 1, it is characterized in that, in described step (3), use one or more proteinase combination in any in trypsase, GluC or Lys-c enzyme-specific to carry out enzymolysis to protein mixture.
7. by method claimed in claim 1, it is characterized in that, in described step (3), adopt Lys-C and Trypsin combination enzymolysis.
8. by method claimed in claim 1, it is characterized in that, in described step (4), adopt the 50cm reverse-phase chromatographic column to be identified; Wherein,
Reversed phase chromatography separation column packing particle diameter is 1.8-5 μ m, and the filler porosity is 10nm-50 nm, and the reversed phase chromatography separation column length is greater than 40cm, and typical length is 50cm, and flow velocity is 100-500 nL/min.
9. by method claimed in claim 1, it is characterized in that, the condition of described three step-length gradient elutions was the 2% acetonitrile 0.1%FA aqueous solution that mobile phase A is pH 2-5, and the 98% acetonitrile 0.1%FA aqueous solution that Mobile phase B is pH 2-5, adopted respectively 0-10%B at 0-30 minute; Adopted respectively the gradient of 10-20%B at 30-240 minute; Adopted respectively the gradient of 20-35%B at 240-340 minute; Within 340-345 minute afterwards, all adopt 35-80%B tonsure, 345-355 minute all to adopt the isocratic elution of 80%B, the gradient that 355-360 minute all adopts 80-0%B; Within 360-400 minute, adopt 0%B to carry out balance to chromatographic column.
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| CN107607642A (en) * | 2017-09-06 | 2018-01-19 | 上海烟草集团有限责任公司 | The multidimensional liquid chromatography mass of albumen and protein groups combination method in a kind of identification tobacco |
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