Impact of digestion conditions on phosphoproteomics

J Proteome Res. 2014 Jun 6;13(6):2761-70. doi: 10.1021/pr401181y. Epub 2014 May 7.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blood Platelets / metabolism
  • Humans
  • Molecular Sequence Data
  • Peptide Mapping
  • Phosphopeptides / chemistry*
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Proteolysis*
  • Proteome / chemistry
  • Proteome / metabolism
  • Solutions
  • Tandem Mass Spectrometry
  • Trypsin / chemistry


  • Phosphopeptides
  • Phosphoproteins
  • Proteome
  • Solutions
  • Trypsin