title: Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag peptides.
abstract: Isotope-coded affinity tag (ICAT) methods, in conjunction with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS), represent a promising approach for accurate protein quantification. However, sensitivity remains a challenge for the quantification of low-copy proteins in complex biological matrices. Here we investigated the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides derivatized with the cleavable ICAT (cICAT) reagent. For cICAT-peptides that were either synthesized or obtained by digestion of model proteins, the cICAT moiety showed a tendency toward protonation under positive ESI, producing relatively intense triply charged cICAT-peptide ions ([IP+3H]3+). [IP+3H]3+ exhibited significantly higher CAD reactivity than did the doubly charged cICAT-peptide ([IP+2H]2+), and produced a greater abundance of fragments at lower collision energies. Fragmentation spectra of [IP+3H]3+ showed variable intensities of doubly charged y and b ions, and the amount of sequence information obtained was dependent on the position of the cICAT-labeled cysteine residue in the peptide sequence. However, the absolute abundances of major fragments of [IP+3H]3+ were much higher than for [IP+2H]2+. Although the efficiency of identification of cICAT-peptides was compromised by their charge distribution toward the triply charged state and by the unique CAD behavior of the [IP+3H]3+ ions, it was found that the triply charged ions provided higher sensitivity than [IP+2H]2+ for quantification using multiple reaction monitoring (MRM). ESI and CAD conditions for MRM of [IP+3H]3+ were optimized, and, for all cICAT-peptides studied, MRM using [IP+3H]3+ as precursors showed 2- to 8-fold higher sensitivity than obtained using [IP+2H]2+, without compromising quantitative accuracy. Using this approach, the time course of tyrosine aminotransferase induction by methylprednisolone was monitored in rat livers. A remarkably better signal-to-noise ratio was observed by using [IP+3H]3+ for quantification compared to [IP+2H]2+.
authors.affiliation: The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, NY 14260-1200, USA. Junqu@Buffalo.edu
authors.forename: Jun
authors.initials: J
authors.lastname: Qu
authors.forename: Robert M
authors.initials: RM
authors.lastname: Straubinger
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008775:Methylprednisolone / Q000494:pharmacology; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D055672:Static Electricity; D014444:Tyrosine Transaminase / Q000032:analysis / Q000378:metabolism*
chemical_list: D011506:Proteins; D014444:Tyrosine Transaminase; D008775:Methylprednisolone