Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.
This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.
Janek
K
K
Institute of Molecular Pharmacology, Robert-Rössle-Str. 10, 13125 Berlin, Germany.
Wenschuh
H
H
Bienert
M
M
Krause
E
E
D002364:Caseins / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002364:Caseins; D010748:Phosphopeptides; D017868:Cyclic AMP-Dependent Protein Kinases