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.
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.
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Authors: Jun Zhong; Min-Sik Kim; Raghothama Chaerkady; Xinyan Wu; Tai-Chung Huang; Derese Getnet; Christopher J Mitchell; Shyam M Palapetta; Jyoti Sharma; Robert N O'Meally; Robert N Cole; Akinori Yoda; Albrecht Moritz; Marc M Loriaux; John Rush; David M Weinstock; Jeffrey W Tyner; Akhilesh Pandey Journal: Mol Cell Proteomics Date: 2012-02-16 Impact factor: 5.911
Authors: Stéphane Bach; Marie Knockaert; Jens Reinhardt; Olivier Lozach; Sophie Schmitt; Blandine Baratte; Marcel Koken; Stephen P Coburn; Lin Tang; Tao Jiang; Dong-Cai Liang; Hervé Galons; Jean-Francois Dierick; Lorenzo A Pinna; Flavio Meggio; Frank Totzke; Christoph Schächtele; Andrea S Lerman; Amancio Carnero; Yongqin Wan; Nathanael Gray; Laurent Meijer Journal: J Biol Chem Date: 2005-06-23 Impact factor: 5.157
Authors: Paturi V Rao; Ashok P Reddy; Xinfang Lu; Surendra Dasari; Adiraju Krishnaprasad; Evan Biggs; Charles T Roberts; Srinivasa R Nagalla Journal: J Proteome Res Date: 2009-01 Impact factor: 4.466