PLDMS: Phosphopeptide Library Dephosphorylation Followed by Mass Spectrometry Analysis to Determine the Specificity of Phosphatases for Dephosphorylation Site Sequences

Methods Mol Biol. 2022;2499:43-64. doi: 10.1007/978-1-0716-2317-6_2.


A detailed understanding of the sequence preference surrounding phosphorylation sites is essential for deciphering the function of the human phosphoproteome . Whereas the mechanisms for substrate site recognition by kinases are relatively well understood, the selection mechanisms for the corresponding phosphatases pose several obstacles. However, multiple pieces of evidence point towards a role of the amino acid sequence in the direct vicinity of the phosphorylation site for recognition by phosphatase enzymes. Peptide library-based studies for enzymes attaching posttranslational modifications (PTMs) are relatively straight forward to carry out. However, studying enzymes removing PTMs pose a challenge in that libraries with a PTM attached are needed as a starting point. Here, we present our methodology using large synthetic phosphopeptide libraries to study the preferred sequence context of protein phosphatases. The approach, termed "phosphopeptide library dephosphorylation followed by mass spectrometry" (PLDMS), allows for the exact control of phosphorylation site incorporation and the synthetic route is capable of covering several thousand peptides in a single tube reaction. Furthermore, it enables the user to analyze MS data tailored to the needs of a specific library and thereby increase data quality. We therefore expect a wide applicability of this technique for a range of enzymes catalyzing the removal of PTMs.

Keywords: Dephosphorylation Assay; Enzyme Kinetics; Mass Spectrometry; Peptide Libraries; Phosphoprotein Phosphatases; Posttranslational Modification; Protein Phosphatases; Protein Phosphorylation; R Analysis; Solid-Phase Peptide Synthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Humans
  • Mass Spectrometry
  • Phosphopeptides* / metabolism
  • Phosphoprotein Phosphatases* / metabolism
  • Phosphorylation
  • Substrate Specificity


  • Phosphopeptides
  • Phosphoprotein Phosphatases