Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry.
Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored.
Parker
Laurie
L
Ludwig Center for Metastasis Research, University of Chicago, Knapp R322, 924 E. 57th Street, Chicago, IL 60637, USA. lparker@uchicago.edu
Engel-Hall
Aaron
A
Drew
Kevin
K
Steinhardt
George
G
Helseth
Donald L
DL
Jabon
David
D
McMurry
Timothy
T
Angulo
David S
DS
Kron
Stephen J
SJ
D002138:Calibration; D008956:Models, Chemical; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D010766:Phosphorylation; D012016:Reference Values; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000592:standards
D010748:Phosphopeptides