Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research

TLDR

iTRAQ reagents are isobaric, so proteins labeled with different tags have identical mass and their proteolytic peptides appear as single peaks in MS scans. The study presents a novel MS‑based method that uses iTRAQ derivatization of primary amino groups for relative protein quantification. The approach labels intact proteins with iTRAQ, separates them by gel electrophoresis, and analyzes the resulting peptides by MS/MS on TOF/TOF or QTOF instruments to generate reporter ions. Reporter ions are efficiently released from singly protonated peptides at low collision energies, while higher energies are required for doubly charged peptides, and the method accurately quantified mixtures of five proteins in varying ratios, demonstrating its general applicability.

Abstract

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.