Monitoring protein kinase and phosphatase reactions with matrix-assisted laser desorption/ionization mass spectrometry and capillary zone electrophoresis: comparison of the detection efficiency of peptide-phosphopeptide mixtures.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary zone electrophoresis (CZE) were evaluated for monitoring protein phosphatase and kinase reactions in vitro. Varying concentrations of peptide C (YIHLEKKYVRRDSG), peptide S (YLIEDNEYTARQGA) and kemptide (LARRSALG) mixed with their corresponding phosphorylated peptides, pC, pS and pkemptide, were analyzed. Comparison between the two techniques indicated that MALDI MS was less quantitative than CZE, showing a bias towards detection of the unphosphorylated peptide S and kemptide. In terms of sensitivity, the MALDI MS and CZE techniques are comparable. Protein kinase A phosphorylation of kemptide was monitored with both MALDI MS and CZE, whereas alkaline phosphatase dephosphorylation of pC could only be monitored with MALDI MS. The absence of inhibition with phosphatase or kinase buffers is a significant advantage of MALDI MS. In contrast to CZE, the MALDI spectra allow identification of the species analyzed by virtue of their mass. The results obtained emphasize the advantage of monitoring enzymatic reactions in buffer solutions using MALDI MS compared with CZE.
Craig
A G
AG
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, San Diego, CA 92138-9216.
Hoeger
C A
CA
Miller
C L
CL
Goedken
T
T
Rivier
J E
JE
Fischer
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004586:Electrophoresis; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D011494:Protein Kinases / Q000032:analysis*
D010748:Phosphopeptides; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases