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  An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples

Parfentev, I., Schilbach, S., Cramer, P., & Urlaub, H. (2020). An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples. Journal of proteomics, 220: 103754. doi:10.1016/j.jprot.2020.103754.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0006-07A9-4 Version Permalink: http://hdl.handle.net/21.11116/0000-0006-07AB-2
Genre: Journal Article

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 Creators:
Parfentev, I.1, Author              
Schilbach, S.2, Author              
Cramer, P.2, Author              
Urlaub, H.1, Author              
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society, ou_578613              
2Department of Molecular Biology, MPI for Biophysical Chemistry, Max Planck Society, ou_1863498              

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Free keywords: Cross-linking mass spectrometry; Peptide database; 20 S proteasome Polymerase II; Transcription factor IIH; Bacillus
 Abstract: Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions. The performance of a parallel XL-MS experiment using a thiol-cleavable cross-linker enabled the identification of peptides that carried a cleaved cross-link moiety after reduction and hence were involved in cross-linking reactions. Based on these identifications, a peptide database was generated and used for the database search of the actual cross-linking experiment with a non-cleavable cross-linker. This peptide-focused approach was tested on protein complexes with a reported structural model and obtained results corresponded well to a conventional database search. An application of the strategy on in vivo cross-linked Bacillus subtilis and Bacillus cereus cells revealed a five- to tenfold reduction in search time and led to significantly more identifications with the latter species than a search against the entire proteome.

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Language(s): eng - English
 Dates: 2020-03-192020-05-30
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.jprot.2020.103754
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Title: Journal of proteomics
Source Genre: Journal
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Pages: - Volume / Issue: 220 Sequence Number: 103754 Start / End Page: - Identifier: -