Tip-Based Fractionation of Batch-Enriched Phosphopeptides Facilitates Easy and Robust Phosphoproteome Analysis

Journal of Proteome Research
Alireza DehghaniDominic Winter

Abstract

The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation to reduce complexity and allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX), and C18 material for basic reversed-phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip-based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide-enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward, reproducible, and requires a fraction of time, effort, and instrumentation compared to thos...Continue Reading

References

Aug 11, 2004·Proceedings of the National Academy of Sciences of the United States of America·Sean A BeausoleilSteven P Gygi
Dec 14, 2004·Nature Biotechnology·John RushMichael J Comb
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Jun 12, 2012·Seminars in Cell & Developmental Biology·Evgeny KanshinPierre Thibault
Oct 24, 2012·Proteomics·Dominic WinterWolf D Lehmann
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Aug 27, 2013·Nature Methods·Hannes HahneBernhard Kuster
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Dec 3, 2016·Nucleic Acids Research·UNKNOWN The Gene Ontology Consortium

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Citations

Oct 1, 2020·The Analyst·Mustafa Gani SürmenNesrin Emekli
Dec 28, 2019·International Journal of Molecular Sciences·Lili DaiPing Zhou
Apr 20, 2018·Journal of the American Society for Mass Spectrometry·Joao A PauloSteven P Gygi

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