Abstract：
:In the past few years, the focus of phosphoproteomics has shifted from merely qualitative to quantitative and targeted studies. Tryptic digestion is a critical step that directly affects quantification and that can be impaired by phosphorylation. Therefore, we systematically characterized the digestion efficiency of 19 nonmodified and phosphorylated model peptides. Whereas we quantified a strong reduction of tryptic cleavage within phosphorylated PKA motifs (R)-R-X-pS/pT and also R-X-X-pT sequences, (R)-R-X-pY sequences were almost unaffected. Structural prediction implied the formation of salt bridges between R/K cleavage sites and phosphoamino acids pS/pT as the main reason for impaired tryptic digestion. We evaluated different conditions to optimize the digestion of such "resistant" phosphopeptides, yielding a substantial improvement of digestion efficiency. We performed a quantitative large-scale phosphoproteomic analysis of human platelets to validate our findings in a complex biological sample. Here, increasing trypsin concentrations up to a trypsin to peptide ratio of 1:10 led to a significant gain (i) in the overall number of phosphorylation sites (up to 9%) and (ii) in the intensities of individual phosphopeptides, thereby improving the sensitivity of phosphopeptide quantification. Still, for certain sequences, the negative impact of phosphorylation on digestion efficiency will further complicate the analysis of phosphorylation stoichiometry.
journal_name
J Proteome Resjournal_title
Journal of proteome researchauthors
Dickhut C,Feldmann I,Lambert J,Zahedi RPdoi
10.1021/pr401181ysubject
Has Abstractpub_date
2014-06-06 00:00:00pages
2761-70issue
6eissn
1535-3893issn
1535-3907journal_volume
13pub_type
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