Novel Concepts of MS-Cleavable Cross-linkers for Improved Peptide Structure Analysis

Abstract

The chemical cross-linking/mass spectrometry (MS) approach is gaining increasing importance as an alternative method for studying protein conformation and for deciphering protein interaction networks. This study is part of our ongoing efforts to develop innovative cross-linking principles for a facile and efficient assignment of cross-linked products. We evaluate two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for automated analysis of cross-linked products. We introduce the bromine phenylurea (BrPU) linker that possesses a unique structure yielding a distinctive fragmentation pattern on collisional activation. Moreover, BrPU delivers the characteristic bromine isotope pattern and mass defect for all cross-linker-decorated fragments. We compare the fragmentation behavior of the BrPU linker with that of our previously described MS-cleavable TEMPO-Bz linker (which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy moiety connected to a benzyl group) that was developed to perform free-radical-initiated peptide sequencing. Comparative collisional activation experiments (collision-induced dissociation and higher-energy collision-induced dissociation) with both cross-linkers were conducted in negative electrospray ionization mode with an Orbitrap Fusion mass spectrometer using five model peptides. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the loss of an intact peptide from the cross-linked products. As the BrPU linker combines a characteristic mass shift with an isotope signature, it presents a more favorable combination for automated assignment of cross-linked products compared with the TEMPO-Bz linker. ᅟ.

An integrated workflow for crosslinking mass spectrometry

“…This permits the sequential digestion workflow to be combined with other crosslinkers such as MS‐cleavable crosslinkers. Recent large‐scale studies have successfully used MS‐cleavable crosslinkers (Liu et al , ; Hage et al , ). Note, however, that our work uses standard crosslinkers at no obvious disadvantage.…”

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…Among them, collision-induced dissociation (CID)-cleavable bonds are most popular as they are present in the majority of existing MS-cleavable reagents. As shown, the two commonly used CID-cleavable bonds are (1) C–S bonds adjacent to sulfoxide, ,,, sulfonium ion, or cyanuric ring; (2) C–N bonds associated with D-P (Asp-Pro) bonds − Rink, ,, urea, ,, or quaternary diamine structures. Depending on the strength of the cleavable bonds, these reagents can be implemented for MS n and/or MS 2 -based workflows to facilitate the identification of cross-linked peptides as detailed in Table .…”

Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology

“…Consequently, considerable effort has been expended on creating crosslinkers that are cleavable in the mass spectrometer so that spectra simply correspond to two modified peptides [94][95][96][97][98][99][100][101][102][103][104][105] .…”