4. Designer Reagents for Mass Spectrometry‐Based Proteomics: Click Chemistry Facilitates Synthesis of Amine‐reactive Multiplexed Isobaric Tags for Protein Quantification

Quantitative mass spectrometry (MS)-based proteomics has been employed to investigate various biological processes by measuring the relative and absolute expression levels of proteins in cells. These studies are enabled by tandem mass spectrometry (MS/MS)-based isobaric tags, made popular by their capability for multiple sample comparison in a single MS experiment. Here, we develop novel isobaric tags for protein quantification, referred to as Caltech Isobaric Tags (CITs), which offer several advantages over other isobaric tags (e.g., iTRAQ and TMT). These include 1) the formation of reporter ions based on a newly discovered low-energy fragmentation pathway, a nucleophilic displacement reaction by the 1,2,3-triazole ring, 2) an unlimited number of isobaric combinations of CIT reagents in principle, 3) an easily tunable reporter ion mass to access clear windows of m/z values not overlapping with peptide MS/MS fragments, and to avoid the low mass–cut off problems inherent in ion trap mass spectrometers, and 4) synthetic methodology that permits preparation of CITs with minimal expense and effort. These advantages are demonstrated by preparing duplex CIT reagents whose reporter ions appear at m/z 164 and 169. CIT reagents are applied to label a model tryptic peptide, protein mixture digests (bovine serum albumin, α and β caseins, ovalbumin, and lysozyme; enolase, aldolase, hemoglobin, creatine kinase, and alcohol dehydrogenase), and Cul1 protein complexes affinitypurified from HEK 293 cells with various ratios. The resulting CIT-labeled peptides are analyzed by either pulsed Q-dissociation (PQD) or higher energy collision dissociation (HCD) in LTQOrbitrap mass spectrometers. Heavy to light ratios of the CIT reporter ions provide excellent