Shotgun identification of protein modifications from protein complexes and lens tissue

Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7900-5. doi: 10.1073/pnas.122231399.


Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylation
  • Amino Acid Sequence
  • CDC2 Protein Kinase / chemistry*
  • CDC2 Protein Kinase / metabolism
  • Cataract / congenital
  • Cataract / metabolism
  • Child, Preschool
  • Crystallins / chemistry*
  • Crystallins / metabolism
  • Humans
  • Lens, Crystalline / chemistry
  • Mass Spectrometry
  • Methylation
  • Molecular Sequence Data
  • Ovalbumin / chemistry*
  • Oxidation-Reduction
  • Phosphorylase a / chemistry*
  • Phosphorylase a / metabolism
  • Phosphorylation
  • Proteome
  • Schizosaccharomyces / chemistry


  • Crystallins
  • Proteome
  • Ovalbumin
  • Phosphorylase a
  • CDC2 Protein Kinase