We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.
- Authors
Ho, Hsin‐Pin; Rathod, Pratikkumar; Louis, Marissa; Tada, Christine K.; Rahaman, Sherida; Mark, Kevin J.; Leng, Jin; Dana, Dibyendu; Kumar, Sanjai; Lichterfeld, Mathias; Chang, Emmanuel J.
- Abstract
RATIONALE Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI-MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times. METHODS A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model. The model is validated for quantifying in vitro phosphorylation assays with inhibition studies on Cdk2/cyclinA. RESULTS Dynamic range of picomoles to femtomoles, good accuracy (deviations of 1.5-3.0% from expected values) and reproducibility (relative standard deviation (RSD) = 4.3-6.3%) are achieved. Inhibition of cyclin-dependent kinase phosphorylation by the classical inhibitors olomoucine and r-roscovitine was evaluated and IC50 values found to be in agreement with reported literature values. These results, achieved with single-point calibration, without isotope or chromatography, compare favorably to those arrived at using isotope dilution ( p >0.5 for accuracy). CONCLUSIONS The mathematical relationship derived here can be applied to a method that we term Double Reciprocal Isotope-free Phosphopeptide Quantification (DRIP-Q), as a strategy for quantification of in vitro phosphorylation assays, the first MALDI-based, isotope- and calibration curve-free method of its type. These results also pave the way for further systematic studies investigating the effect of peptide composition and experimental conditions on quantitative, label-free MALDI. Copyright © 2014 John Wiley & Sons, Ltd.
- Subjects
MATRIX-assisted laser desorption-ionization; PHOSPHOPEPTIDES; MASS spectrometry; CHROMATOGRAPHIC analysis; PHOSPHORYLATION
- Publication
Rapid Communications in Mass Spectrometry: RCM, 2014, Vol 28, Issue 24, p2681
- ISSN
0951-4198
- Publication type
Article
- DOI
10.1002/rcm.7063