Targeting proline in (phospho)proteomics

FEBS J. 2020 Jul;287(14):2979-2997. doi: 10.1111/febs.15190. Epub 2020 Jan 13.


Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C-terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline-directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications.

Keywords: (phospho)proteomics; EndoPro; mass spectrometry; proline effect; protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • HeLa Cells
  • Humans
  • Neoplasm Proteins / metabolism*
  • Peptide Hydrolases / metabolism*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proline / metabolism*
  • Proteolysis
  • Proteome / analysis
  • Proteome / metabolism*
  • Proteomics / methods*
  • Trypsin / metabolism*


  • Neoplasm Proteins
  • Phosphoproteins
  • Proteome
  • Proline
  • Peptide Hydrolases
  • Trypsin