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J Proteome Res. 2019 Mar 1;18(3):1363-1370. doi: 10.1021/acs.jproteome.8b00947. Epub 2019 Feb 18.

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

Author information

1
Institute of Molecular Pathology (IMP) , Vienna BioCenter (VBC) , Vienna 1030 , Austria.
2
Institute of Molecular Biotechnology (IMBA), Austrian Academy of Sciences , Vienna BioCenter (VBC) , Vienna 1030 , Austria.
3
Gregor Mendel Institute (GMI), Austrian Academy of Sciences , Vienna BioCenter (VBC) , Vienna 1030 , Austria.

Abstract

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 to obtain the best-quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone are yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl-sulfoxide-based cross-links on HCD-equipped mass spectrometers. From the comparison of nine 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-enabled (MSn) instrumentation and alternative dissociation techniques. Data are available via ProteomeXchange with identifier PXD011861.

KEYWORDS:

DSSO; XLMS; cleavable cross-linker; cross-linking mass spectrometry; stepped HCD

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