Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis

Anal Chem. 2005 Aug 15;77(16):5243-50. doi: 10.1021/ac050232m.


We have developed a multi-protease approach that allows sensitive and comprehensive mapping of protein phosphorylation sites. The combined application of the low-specificity proteases elastase, proteinase K, and thermolysin in addition to trypsin results in high sequence coverage, a prerequisite for comprehensive phosphorylation site mapping. Phosphopeptide enrichment is performed with the recently introduced phosphopeptide affinity material titansphere. We have optimized the selectivity of the phosphopeptide enrichment with titansphere, without compromising the high recovery rate of approximately 90%. Phosphopeptide-enriched fractions are analyzed with a highly sensitive nanoLC-MS/MS system using a 25-microm-i.d. reversed-phase column, operated at a flow rate of 25 nL/min. The new approach was applied to the murine circadian protein period 2 (mPER2). A total of 21 phosphorylation sites of mPER2 have been detected by the multi-protease approach, whereas only 6 phosphorylation sites were identified using solely trypsin. Titansphere proved to be well suited for the enrichment of a large variety of phosphopeptides, including peptides carrying two, three, or four phosphorylated residues, as well as phosphopeptides containing more basic than acidic amino acids.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Cycle Proteins / analysis*
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism
  • Chromatography, Liquid / methods*
  • Mice
  • Molecular Sequence Data
  • Nanotechnology / methods*
  • Nuclear Proteins / analysis*
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Peptide Hydrolases / metabolism
  • Period Circadian Proteins
  • Phosphopeptides / analysis*
  • Phosphopeptides / chemistry*
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Tandem Mass Spectrometry / methods*
  • Transcription Factors / analysis*
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism


  • Cell Cycle Proteins
  • Nuclear Proteins
  • Per2 protein, mouse
  • Period Circadian Proteins
  • Phosphopeptides
  • Transcription Factors
  • Peptide Hydrolases