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

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.