A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS)

Abstract

The chemical cross-linking/mass spectrometry (MS) approach is a growing research field in structural proteomics that allows gaining insights into protein conformations. It relies on creating distance constraints between cross-linked amino acid side chains that can further be used to derive protein structures. Currently, the most urgent task for designing novel cross-linking principles is an unambiguous and automated assignment of the created cross-linked products. Here, we introduce the homobifunctional, amine-reactive, and water soluble cross-linker azobisimidoester (ABI) as a prototype of a novel class of cross-linkers. The ABI-linker possesses an innovative modular scaffold combining the benefits of collisional activation lability with open shell chemistry. This MS-cleavable cross-linker can be efficiently operated via free radical initiated peptide sequencing (FRIPS) in positive ionization mode. Our proof-of-principle study challenges the gas phase behavior of the ABI-linker for the three amino acids, lysine, leucine, and isoleucine, as well as the model peptide thymopentin. The isomeric amino acids leucine and isoleucine could be discriminated by their characteristic side chain fragments. Collisional activation experiments were conducted via positive electrospray ionization (ESI) on two Orbitrap mass spectrometers. The ABI-mediated formation of odd electron product ions in MS/MS and MS experiments was evaluated and compared with a previously described azo-based cross-linker. All cross-linked products were amenable to automated analysis by the MeroX software, underlining the future potential of the ABI-linker for structural proteomics studies. Graphical Abstract ᅟ.

Free radical‐initiated peptide sequencing (FRIPS)‐based cross‐linkers for improved peptide and protein structure analysis

“…HCD product ion mass spectra of (upper panel) 3+ and (lower panel) 4+ charged ions of ABI‐linked thymopentin (RKDVY); spectra were recorded at 30% NCE. Product ions were automatically assigned with the MeroX software …”

“…Recently, a complementary homobifunctional, symmetric, amine‐reactive, and water‐soluble FRIPS cross‐linker azobisimidoester (ABI) was introduced, possessing the additional advantage that it can be efficiently operated in (+)ESI experiments (Figure ). The structure of the ABI reagent is an optimized design of the azo‐reagents for FRIPS discussed above, which all have the initial CID triggered N 2 loss in common, generating peptide radicals for FRIPS.…”

“…Reactivity of the ABI‐linker with amine groups in peptides; nomenclature of the fragments generated in (+)ESI upon collisional activation (CID and HCD) …”

“…Proposed diagnostic fragmentation pattern of doubly protonated ion of cross‐linked leucine at m/z 214.156 …”

“…Spectra were recorded at 30% NCE. Please note that the proposed structures are putative; proposed fragmentation patterns of the doubly protonated ion of cross‐linked leucine are presented in Figure …”