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.
CITATION STYLE
Zaman, K., Pandey, P., Shulaev, V., & Prokai, L. (2022). Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionation Using Solid-Phase Extraction Cartridges. In Methods in Molecular Biology (Vol. 2396, pp. 71–84). Humana Press Inc. https://doi.org/10.1007/978-1-0716-1822-6_7
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