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RP-SAX-RP platform-based proteome sequence analysis method

A technology of RP-SAX-RP, 3DRP-SAX-RP, applied in the field of analysis, can solve problems such as limiting total protein sequence coverage

Inactive Publication Date: 2018-07-20
上海悦良生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These observations and results ultimately limit the resolution peak capacity and total protein sequence coverage that can be obtained from 2-D platforms

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A proteome sequence analysis method based on the RP-SAX-RP platform, including the construction of a 3D RP-SAX-RP system; comprising the following steps:

[0018] (1) 3D RP-SAX-RP platform includes Waters UPLC, isocratic pump, autosampler (Waters Corp., Milford, MA) and additional two-position six-port switching valve (Valco Inc., Austin, TX);

[0019] (2) One-dimensional reversed-phase column including a 250 μm inner diameter, 15 cm long capillary filled with 5 μm diameter XBridge C18 resin (Waters Corp., Milford, MA), SAX column (10 μm diameter POROS10HQ resin filled with 250 μm inner diameter, 15cm long capillary) is directly connected to the discharge port of the one-dimensional RP column;

[0020] (3) An isocratic pump passes through the sampling loop at a speed of 2 μL / min to deliver the peptide sample or one-dimensional (acetonitrile in 20 mM carbamic acid, pH 10) and two-dimensional (KCl in 20 mM carbamic acid, pH 10) ) eluent;

[0021] (4) The binary pump del...

Embodiment 2

[0025] A detailed comparison of automated, online 2D RP-RP and SCX-RP fractionation platforms for the analysis of peptides from complex cell lysates and affinity-purified multicomponent protein complexes; given reversed-phase The high degree of orthogonality of the separation, suggests that the additional addition of three-dimensional separation may significantly increase the separation efficiency; based on the buffer conditions applied on the RP-RP one-dimensional platform (20 mM carbamic acid, pH 10), in a 3D RP-SAX-RP configuration The strong anion exchange is set to two-dimensional; in simple terms, peptides are loaded in carbamate buffer through an autosampler and intercepted on a one-dimensional RP column; peptides that are difficult to retain on reversed-phase columns at high pH will fall into Two-dimensional (SAX, pH 10) or three-dimensional (RP, Ph3.0) columns; the low-pH precolumn and analytical column constitute a three-dimensional fractionation, installed in an open...

Embodiment 3

[0027]Previous analytical characterization studies showed that RP-RP performance exhibited mass spectral peak depths between 30-40 fragments using LC-MS / MS for the analysis of E. coli peptides in a complex background of total cell lysates; based on these observations Asked whether PR-SAX-RP could improve the depth of classification and exceed the performance of diminishing returns previously exhibited by RP-RP; to this end, RP-RP (40 fragments) and RP-SAX were repeatedly performed on E. coli peptides - RP (37 fragments) analysis; results showed that although each fractionation technique provided reproducibility data, the RP-SAX-RP platform identified 44% and 31% more peptides and proteins than RP-RP; Alternative analysis of peak volume, next suggested whether the number of fragments identifying discrete peptides varied between the two binning platforms; in fact, 75% of all peptide identifications spanned a single RP-SAX-RP fragment, whereas RP-RP was reduced to 60%; consistent...

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PUM

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Abstract

The invention discloses an RP-SAX-RP platform-based proteome sequence analysis method. According to the method, separation means based on an automatic nano upgrading flow velocity 3DLC-MS / MS platformof high PH reverse phase-intense anion exchange-low PH reverse phase are adopted to analyze complex proteome; when tryptic peptide from escherichia coli is analyzed, RP-SAX-RP is prior to an RP-RP technique, in addition, identification that each brewer's yeast cell has 50 copy level proteins can be achieved, and detection limit of 500amol of 40mu g of total lysate of a low-resolution 3-D ion trapmass spectrometer is correspondingly achieved; similar research is carried out on LTQ-Orbitrap, under absorption and analysis of single 101fractionRP-SAX-RPLC-MS / MS, 4000 specific proteins are generated from 5mu g of the yeast lysate, and an upper limit about 65amol is provided for protein expression of a copy number 50 of each cell.

Description

technical field [0001] The invention relates to an analysis method, in particular to a proteome sequence analysis method based on the RP-SAX-RP platform. Background technique [0002] Mass spectrometry-based proteomics has been validated to characterize proteins in a wide range of experimental settings: recombinant protein expression systems, protein analysis by SDS-PAGE resolution, isolation of multicomponent protein complexes by affinity purification, post-transcriptional modification (phospho glycosylation, sulfation, ubiquitination and ubiquitination), protein subdomains and terminals, subcellular compartments and finally the entire proteome. Improvements in instrumentation continue to increase the application and development of mass spectrometry in the biomedical field, however the fact is that data quality often varies inversely with sample complexity. Detrimental effects observed in peptide analysis appear in composite models, both exhibiting reduced performance in M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/89G01N27/62
CPCG01N30/89G01N33/6848
Inventor 戚良
Owner 上海悦良生物科技有限公司