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

ABSTRACT: 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 reduces 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 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 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 multistage MS (MSn) enabled instrumentation and alternative dissociation techniques.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Equus Caballus (horse) Homo Sapiens (human) Bos Taurus (bovine) Escherichia Coli Streptococcus Pyogenes Serotype M2 Cicer Arietinum (chickpea) (garbanzo) Oryctolagus Cuniculus (rabbit) Candida Albicans (yeast)

SUBMITTER: Christian Stieger  

LAB HEAD: Karl Mechtler

PROVIDER: PXD011861 | Pride | 2019-02-01

REPOSITORIES: Pride