Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.
BACKGROUND
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.
Department of Chemistry, The Graduate Center, The City University of New York, New York, NY, USA; Department of Chemistry, York College, The City University of New York, Jamaica, NY, USA.
Hsin-Pin
HP
Ho
Pratikkumar
P
Rathod
Marissa
M
Louis
Christine K
CK
Tada
Sherida
S
Rahaman
Kevin J
KJ
Mark
Jin
J
Leng
Dibyendu
D
Dana
Sanjai
S
Kumar
Mathias
M
Lichterfeld
Emmanuel J
EJ
Chang
D000595:Amino Acid Sequence; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019925:Cyclin A / Q000037:antagonists & inhibitors / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D016014:Linear Models; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D019925:Cyclin A; D010748:Phosphopeptides; D051357:Cyclin-Dependent Kinase 2