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Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC–MS/MS

  • Marina A. Gritsenko
  • Zhe Xu
  • Tao LiuEmail author
  • Richard D. SmithEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)

Abstract

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC–MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

Key words

Quantitative proteomics Isobaric labeling iTRAQ Two-dimensional liquid chromatography Mass spectrometry 

Notes

Acknowledgments

Portions of this work were supported by the grant U24CA160019, from the National Cancer Institute Clinical Proteomic Tumor Analysis Consortium (CPTAC) and National Institutes of Health grant P41GM103493. The experimental work described herein was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE and located at Pacific Northwest National Laboratory, which is operated by Battelle Memorial Institute for the DOE under Contract DE-AC05-76RL0 1830.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Biological Sciences Division and Environmental Molecular Sciences LaboratoryPacific Northwest National LaboratoryRichlandUSA

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