Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.
Chen
J
J
Division of Hematology/Oncology, Vanderbilt University, Nashville, Tennessee 37232-6305, USA.
Qi
Y
Y
Zhao
R
R
Zhou
G W
GW
Zhao
Z J
ZJ
D009842:Oligopeptides / Q000032:analysis; D010748:Phosphopeptides / Q000737:chemistry; D017027:Protein Tyrosine Phosphatases / Q000032:analysis*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
D009842:Oligopeptides; D010748:Phosphopeptides; C081990:glutamyl-asparaginyl-aspartyl-tyrosyl-isoleucyl-asparaginyl-alanyl-seryl-leucine; D017027:Protein Tyrosine Phosphatases