Unlock instant, AI-driven research and patent intelligence for your innovation.

A method for the separation and identification of proteomes based on one-dimensional long-column liquid chromatography-tandem mass spectrometry

A technology of liquid chromatography and tandem mass spectrometry, which is applied in the field of fast and efficient proteome separation and identification based on one-dimensional long-column liquid chromatography-tandem mass spectrometry, can solve the problems of large sample consumption, time-consuming, cumbersome operation, etc., and achieve experimental operation Simple, easy to operate, simple steps effect

Inactive Publication Date: 2015-10-07
FUDAN UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to improve the identification rate and increase the coverage of proteins, some studies have proposed a series of two-dimensional separation methods; in research practice, combined methods are often used, such as SDS-PAGE and RPLC, IEF and RPLC, SAX / SCX and RPLC, etc. The identification of proteins can be improved through the combination of different separation methods, but the two-dimensional separation method takes a long time, consumes a large amount of samples, and is cumbersome to operate;

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for the separation and identification of proteomes based on one-dimensional long-column liquid chromatography-tandem mass spectrometry
  • A method for the separation and identification of proteomes based on one-dimensional long-column liquid chromatography-tandem mass spectrometry
  • A method for the separation and identification of proteomes based on one-dimensional long-column liquid chromatography-tandem mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] HepG2 cells were cultured by our laboratory; HepG2 cells were washed and suspended in 100mM TrisHCl pH 7.6 lysate containing SDS, DTT and protease inhibitors, incubated at 95°C for 5min, cooled to room temperature, briefly sonicated, and centrifuged to take the supernatant , methanol chloroform precipitation, 100mM TrisHCl / 6M urea redissolved the protein, and then used Lys-C and trypsin for enzymatic hydrolysis to obtain its peptide mixture.

[0039]After enzymolysis of 100 μg protein mixture extracted from HepG2 cells, 2 μg of mixed peptides were taken for one-dimensional reversed-phase chromatography separation-electrospray tandem mass spectrometry data acquisition and analysis; the mobile phase A used for low pH reversed-phase chromatography separation was 0.1% Aqueous solution of 2% formic acid in acetonitrile, mobile phase B is an aqueous solution of 0.1% formic acid and 98% acetonitrile; the particle size of the reversed-phase chromatography separation column packi...

Embodiment 2

[0041] HUVEC whole protein lysate was provided by C-HPP. Methanol chloroform precipitation, 100mM TrisHCl / 6M urea redissolved the protein, and then enzymatically digested it with Lys-C and trypsin to obtain its peptide mixture.

[0042] After enzymolysis of 100 μg protein mixture of HUVEC whole protein extract, 4 μg of mixed peptides were taken for one-dimensional reversed-phase chromatography separation-electrospray tandem mass spectrometry data collection and analysis; the mobile phase A used for low pH reversed-phase chromatography separation was An aqueous solution of 0.1% formic acid and 2% acetonitrile, mobile phase B is an aqueous solution of 0.1% formic acid and 98% acetonitrile; the particle size of the reverse-phase chromatography separation column packing is 2 μm, the filling diameter is 10 nm, and the reverse-phase chromatography separation column is 75 μm×500 mm; The flow rate is 200 nL / min. According to the gradient setting, 0-10% B is used at 0-30 minutes; 10-2...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
lengthaaaaaaaaaa
particle diameteraaaaaaaaaa
porosityaaaaaaaaaa
Login to View More

Abstract

The invention belongs to the field of biological technology, and relates to a proteome separation and identification method based on one-dimensional long-column liquid chromatography tandem mass spectrometry. The identification method uses a 50cm reversed-phase chromatographic column and uses a one-dimensional long gradient to separate the proteomics samples. After electrospray ionization and tandem mass spectrometry, the one-dimensional proteomics is separated and identified. The results show that the experimental operation of this identification method is simple. , No need for pre-fractionation, one experiment can achieve the effect of traditional two-dimensional separation mass spectrometry identification; especially for the processing of a small amount of samples, one experiment can complete a proteomics data analysis with only a few micrograms of samples, and the required sample volume Less, the instrument analysis efficiency of complex biological proteomes can achieve the analysis and identification of more than 4,000 proteins within 7 hours, and realize the rapid and efficient identification of proteins.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for separating and identifying proteomes, in particular to a method for rapidly and efficiently separating and identifying proteomes based on one-dimensional long-column liquid chromatography tandem mass spectrometry. Background technique [0002] The prior art discloses that proteomics is the science of studying all the proteins expressed in a genome. It was first proposed by Marc Wilkins in the early 1990s. It is a large-scale study of proteins, including identification of proteins, post-translational modifications of proteins and their interaction etc. It is known that the composition of proteomic biological samples is complex and needs to be separated before detection to reduce the complexity of the sample. The commonly used methods are sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), Isoelectric focusing electrophoresis (IEF), strong anion / c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/89
Inventor 杨芃原陆豪杰殷薛飞刘晓慧申华莉晏国全陈晨
Owner FUDAN UNIV