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通过结合 PAGE 和肽等电聚焦提高蛋白质组覆盖度：基于凝胶分离方法的比较研究。

Increased proteome coverage by combining PAGE and peptide isoelectric focusing: comparative study of gel-based separation approaches.

机构信息

Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

出版信息

Proteomics. 2013 Oct;13(20):2947-55. doi: 10.1002/pmic.201300035.

DOI:10.1002/pmic.201300035
PMID:23943586
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4285200/
Abstract

The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.

摘要

在 shotgun 蛋白质组学实验中，对复杂蛋白质组样品进行深入分析需要将样品分成亚样品进行 LC-MS/MS 分析。我们已经建立了一种用于 shotgun 蛋白质组学的 3D 工作流程，该流程依赖于 1D PAGE、胶分馏、胰蛋白酶消化以及胶内 IEF 进行肽分离，然后进行 RP-HPLC-MS/MS。我们的结果表明，应用肽 IEF 可以显著增加从 PAGE 亚分级中鉴定出的蛋白质数量。该方法提供了更深层次的蛋白质组覆盖度，并通过仍然依赖于第一维分子量的蛋白质分离，在实验上处理高度复杂混合物时提供了很大的灵活性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/b6e08a7ad2f2/pmic0013-2947-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/6e7dfc76515d/pmic0013-2947-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/3101a6c2a340/pmic0013-2947-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/b280173dd46a/pmic0013-2947-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/b6e08a7ad2f2/pmic0013-2947-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/6e7dfc76515d/pmic0013-2947-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/3101a6c2a340/pmic0013-2947-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/b280173dd46a/pmic0013-2947-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cdc/4285200/b6e08a7ad2f2/pmic0013-2947-f4.jpg

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