Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

Protein
phosphorylation is an important post-translational modification
(PTM) involved in embryonic development, adult homeostasis, and disease.
Over the past decade, several advances have been made in liquid chromatography–tandem
mass spectrometry (LC–MS/MS)-based technologies to identify
thousands of phosphorylation sites. However, in-depth phosphoproteomics
often require off-line enrichment and fractionation techniques. In
this study, we provide a detailed analysis of the physicochemical
characteristics of phosphopeptides, which have been fractionated by
off-line high-pH chromatography (HpH) before subsequent titanium dioxide
(TiO2) enrichment and LC–MS/MS analysis. Our results
demonstrate that HpH is superior to standard strong-cation exchange
(SCX) fractionation in the total number of phosphopeptides detected
when analyzing the same number of fractions by identical LC–MS/MS
gradients. From 14 HpH fractions, we routinely identified over 30 000
unique phosphopeptide variants, which is more than twice the number
of that obtained from SCX fractionation. HpH chromatography displayed
an exceptional ability to fractionate singly phosphorylated peptides,
with minor benefits for doubly phosphorylated peptides over that with
SCX. Further optimizations in the pooling and concatenation strategy
increased the total number of multiphosphorylated peptides detected
after HpH fractionation. In conclusion, we provide a basic framework
and resource for performing in-depth phosphoproteome studies utilizing
off-line basic reversed-phased fractionation. Raw data is available
at ProteomeXchange (PXD001404).