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Published February 8, 2012 | public
Journal Article Open

Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification


We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d_5-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Additional Information

© 2012 American Chemical Society. Received: October 20, 2011. Just Accepted Manuscript January 05, 2012. Publication Date (Web): January 5, 2012. This work was supported by the National Science Foundation (NSF) through grant CHE-0416381 (J.L.B.), the Beckman Institute at California Institute of Technology (J.L.B., M.J.S., S.H., and R.J.L.G.), and the National Institutes of Health (NIH) through grant RR 20004 (J.A.L.), and the Betty and Gordon Moore Foundation (S.H. and R.J.L.G). Computational resources for DFT results were kindly provided by the Materials and Process Simulation Center at California Institute of Technology. C.H.S. acknowledges a fellowship from the Kwanjeong Educational Foundation. J.E.L. was supported by the Ruth L. Kirschstein NRSA fellowship from the NIH (CA138126).

Attached Files

Supplemental Material - ja2099003_si_001.pdf

Accepted Version - nihms354213.pdf


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August 19, 2023
August 19, 2023