Selective Enrichment of Cysteine-Containing Phosphopeptides for Subphosphoproteome Analysis

Among
the natural amino acids, cysteine is unique since it can
form a disulfide bond through oxidation and reduction of sulfhydryl
and thus plays a pervasive role in modulation of proteins activities
and structures. Crosstalk between phosphorylation and other post-translational
modifications has become a recurrent theme in cell signaling regulation.
However, the crosstalk between the phosphorylation and the formation
and reductive cleavage of disulfide bond has not been investigated
so far. To facilitate the study of this crosstalk, it is important
to explore the subset of phosphoproteome where phosphorylations are
occurred near to cysteine in the protein sequences. In this study,
we developed a straightforward sequential enrichment method by combining
the thiol affinity chromatography with the immobilized titanium ion
affinity chromatography to selectively enrich cysteine-containing
phosphopeptides. The high specificity and high sensitivity of this
method were demonstrated by analyzing the samples of Jurkat cells.
This “divide and conquer” strategy by specific analysis
of a subphosphoproteome enables identification of more low abundant
phosphosites than the conventional global phosphoproteome approach.
Interestingly, amino acid residues surrounding the identified phosphosites
were enriched with buried residues (L, V, A, C) while depleted with
exposed residues (D, E, R, K). Also, the phosphosites identified by
this approach showed a dramatic decrease in locating in disorder regions
compared to that identified by conventional global phosphoproteome.
Further analysis showed that more proline directed kinases and fewer
acidophilic kinases were responsible for the phosphorylation sites
of this subphosphoproteome.