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Optimized fragmentation improves the identification of peptides cross-linked using MS-cleavable reagents

By Christian E. Stieger, Philipp Doppler, Karl Mechtler

Posted 23 Nov 2018
bioRxiv DOI: 10.1101/476051 (published DOI: 10.1021/acs.jproteome.8b00947)

Cross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometry-cleavable (MS-cleavable) reagents dramatically reduce complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized the MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)-based cross-links on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped-HCD fragmentation methods that were evaluated on a variety of cross-linked model proteins. The optimal stepped-HCD-method was then directly compared with previously described methods using an Orbitrap Fusion Lumos Tribrid instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multi-stage MS (MSn) enabled instrumentation and alternative dissociation techniques.

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