Quantitative analysis of modified proteins by LC-MS/MS of peptides labeled with phenyl isocyanate

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 d(0)- and d(5)-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 d(0)- and d(5)-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.