Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionation Using Solid-Phase Extraction Cartridges

Khadiza Zaman, Prajita Pandey, Vladimir Shulaev, Laszlo Prokai

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Discovery-driven comparative proteomics employing the bottom-up strategy with label-free quantification on high-resolution mass analyzers like an Orbitrap in a hybrid instrument has the capacity to reveal unique biological processes in the context of plant metabolic engineering. However, proteins are very heterogeneous in nature with a wide range of expression levels, and overall coverage may be suboptimal regarding both the number of protein identifications and sequence coverage of the identified proteins using conventional data-dependent acquisitions without sample fractionation before online nanoflow liquid chromatography–mass spectrometry (LC–MS) and tandem mass spectrometry (MS/MS). In this chapter, we detail a simple and robust method employing high-pH reversed-phase (HRP) peptide fractionation using solid-phase extraction cartridges for label-free proteomic analyses. Albeit HRP fractionation separates peptides according to their hydrophobicity like the subsequent nanoflow gradient reversed-phased LC relying on low pH mobile phase, the two methods are orthogonal. Presented here as a protocol with yeast (Saccharomyces cerevisiae) as a frequently used model organism and hydrogen peroxide to exert cellular stress and survey its impact compared to unstressed control as an example, the described workflow can be adapted to a wide range of proteome samples for applications to plant metabolic engineering research.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages71-84
Number of pages14
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2396
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • High pH reversed phase
  • Label-free quantitation
  • LC-MS/MS
  • Peptide fractionation
  • Proteomics
  • Solid-phase extraction
  • Yeast (Saccharomyces cerevisiae)

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