Improve the Coverage for the Analysis of Phosphoproteome of HeLa Cells by a Tandem Digestion Approach

Complete coverage of all phosphorylation sites in a proteome
is
the ultimate goal for large-scale phosphoproteome analysis. However,
only making use of one protease trypsin for protein digestion cannot
cover all phosphorylation sites, because not all tryptic phosphopeptides
are detectable in MS. To further increase the phosphoproteomics coverage
of HeLa cells, we proposed a tandem digestion approach by using two
different proteases. By combining the data set of the first Glu-C
digestion and the second trypsin digestion, the tandem digestion approach
resulted in the identification of 8062 unique phosphopeptides and
8507 phosphorylation sites in HeLa cells. The conventional trypsin
digestion approach resulted in the identification of 3891 unique phosphopeptides
and 4647 phosphorylation sites. It was found that the phosphorylation
sites identified from the above two approaches were highly complementary.
By combining above two data sets, in total we identified 10899 unique
phosphopeptides and 11262 phosphorylation sites, corresponding to
3437 unique phosphoproteins with FDR < 1% at peptide level. We
also compared the kinase motifs extracted from trypsin, Glu-C, or
a second trypsin digestion data sets. It was observed that basophilic
motifs were more frequently found in the trypsin and the second trypsin
digestion data sets, and the acidic motifs were more frequently found
in the Glu-C digestion data set. These results demonstrated that our
tandem digestion approach is a good complement to the conventional
trypsin digestion approach for improving the phosphoproteomics analysis
coverage of HeLa cells.