Phosphoprotein Analysis by MALDI-TOF Mass Spectrometry using On-Probe Tandem Proteolysis and Dephosphorylation

Agron Mataj, Reinhard I. Boysen, Milton T.W. Hearn

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In this investigation, methods based on on-probe enzymatic cleavage matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analyses have been developed, allowing the rapid assignment of phosphorylation sites within phosphoproteins. The procedures involved robotic sample deposition of a phosphoprotein, such as intact bovine β-casein, on stainless steel or gold MALDI plates, on-probe proteolysis with trypsin for 10–180 s at 37°C, on-probe dephosphorylation for 1–10 min at 37°C with alkaline phosphatase, followed by differential mass spectrometry with peptide mass mapping. The dephosphorylation conditions were initially optimized using in-solution tryptic digestion of the phosphoprotein performed in the presence of MS-compatible anionic surfactant sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate. Two methods of trypsin deactivation were investigated, cooling and quenching by acidification, which resulted in the surfactant either staying intact or becoming cleaved, respectively. Since the surfactant had no detrimental effects on dephosphorylation of phosphopeptides, the acidification and neutralization steps were not included in the final analytical method. A protocol, comprising on-probe tandem, surfactant-aided proteolysis for 3 min followed by on-probe dephosphorylation for 10 min was thus established, allowing the rapid identification of location and sequence of phosphopeptides within a phosphoprotein by these procedures.

Original languageEnglish
Pages (from-to)1463-1482
Number of pages20
JournalAnalytical Letters
Issue number9
Publication statusPublished - 13 Jun 2017


  • acid labile surfactant
  • alkaline phosphatase
  • dephosphorylation
  • matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • phosphoproteins
  • post-translational modification
  • trypsin
  • β-Casein

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