Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line

Phosphoproteomics
requires better separation of phosphopeptides
to boost the coverage of the phosphoproteome. We argue that an alternative
separation method that produces orthogonal phosphopeptide separation
to the widely used LC needs to be considered. Capillary zone electrophoresis
(CZE) is one important alternative because CZE and LC are orthogonal
for phosphopeptide separation and because the migration time of peptides
in CZE can be accurately predicted. In this work, we coupled strong
cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale
phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based
platform identified 11,555 phosphopeptides. The phosphopeptide data
set is at least 100% larger than that from previous CZE-MS/MS studies
and will be a valuable resource for building a model for predicting
the migration time of phosphopeptides in CZE. Phosphopeptides migrate
significantly slower than corresponding unphosphopeptides under acidic
conditions of CZE separations and in a normal polarity. According
to our modeling data, phosphorylation decreases peptide’s charge
roughly by one charge unit, resulting in dramatic decrease in electrophoretic
mobility. Preliminary investigations demonstrate that electrophoretic
mobility of phosphopeptides containing one phosphoryl group can be
predicted with the same accuracy as for nonmodified peptides (R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS
were complementary in large-scale phosphopeptide identifications and
produced different phosphosite motifs from the HCT116 cell line. The
data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary
separation approach for not only improving the phosphoproteome coverage
but also providing more insight into the phosphosite motifs.