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

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 those of the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.