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 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 10000-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.
Mason
Daniel E
DE
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721-0207, USA.
Liebler
Daniel C
DC
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004852:Epoxy Compounds / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry; D017953:Isocyanates; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000032:analysis*; D013194:Staining and Labeling
D004852:Epoxy Compounds; D006454:Hemoglobins; D017953:Isocyanates; D010455:Peptides; C025319:phenyl isocyanate; C013690:styrene oxide