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⁺. Copyright © 2005 John Wiley & Sons, Ltd.