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Studies on quantitative phosphopeptide analysis by matrix‐assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves

Year: 2014
Type: article
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.
DOI: 10.1002/rcm.7063
Authors: Hsin‐Pin Ho, Pratikkumar Rathod, Marissa Louis, Christine K. Tada, Sherida Rahaman, Kevin J. Mark, Jin Leng, Dibyendu Dana, Sanjai Kumar, Mathias Lichterfeld, Emmanuel J. Chang
Institutions: The Graduate Center, CUNY, York College, City University of New York, City University of New York, LaGuardia Community College, Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital, Massachusetts Institute of Technology, Queens College, CUNY
Language: English
Subfield: Spectroscopy
Field: Chemistry
Open Access status: green
Funders: Doris Duke Charitable Foundation, amfAR, The Foundation for AIDS Research, City University of New York, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases
Awards: Innate resistance of CD4 T cells to HIV-1 infection in elite controllers, G297103590, G4986927018, G5049575800, G8259407239, Using stable isotope labeling/mass spectrometry to probe Cdk1 multisite phosphory, G8632243, Inhibitors of host kinases as molecular targets for immune defense against HIV-1
OpenAlex ID: W1846619136
FWCI: 0.571
Cites: 32
Cited by: 8
Related to: 10