Proteomics

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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): Escherichia coli   Cicer arietinum   Candida albicans   Homo sapiens   Equus caballus   Bos taurus   Oryctolagus cuniculus   Streptococcus pyogenes serotype M2  

TISSUE(S): Tissue Not Applicable To Dataset

DISEASE(S): Not Available

SUBMITTER: Christian Stieger  

LAB HEAD: Karl Mechtler

PROVIDER: PXD011861 | Pride | 2019-02-01

REPOSITORIES: pride

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Publications

Optimized Fragmentation Improves the Identification of Peptides Cross-Linked by MS-Cleavable Reagents.

Stieger Christian E CE   Doppler Philipp P   Mechtler Karl K  

Journal of proteome research 20190218 3


Cross-linking mass spectrometry is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by nonspecialist laboratories. Specifically, the use of novel mass-spectrometry-cleavable (MS-cleavable) reagents dramatically reduces the 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  ...[more]

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