Quantitative Analysis of Modified Proteins by LC−MS/MS of Peptides Labeled with Phenyl Isocyanate
- 22 January 2003
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 2 (3), 265-272
- https://doi.org/10.1021/pr0255856
Abstract
Stable isotope tagging methods have enabled relative quantitation of proteins between samples in LC−MS/MS analyses. However, most such methods are not applicable to the differential quantitation of modified proteins because the isotope tagging reagents only react with certain peptides or because the reagents incorporate a mass increment that is too small to allow reliable quantitation on low resolution ion trap MS instruments. Here, we describe the use of d0- and d5-phenyl isocyanate (PIC) as N-terminal reactive tags for essentially all peptides in proteolytic digests. PIC reacts quantitatively with peptide N-terminal amines within minutes at neutral pH and the PIC-labeled peptides undergo informative MS/MS fragmentation. Ratios of d0- and d5-PIC-labeled derivatives of several model peptides were linear across a 10 000-fold range of peptide concentration ratios, thus indicating a wide dynamic range for quantitation. Application of PIC labeling enabled relative quantitation of several styrene oxide adducts of human hemoglobin in LC−MS/MS analyses. PIC labeling offers a versatile means of quantifying changes in modified or variant protein forms in paired samples. Keywords: adducts • isotope tag • phenyl isocyanate • quantitation • tandem mass spectrometryKeywords
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