Proteomics

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

ABSTRACT: The application of non-cleavable cross-linkers to complex samples in XL-MS workflows is limited by the n² problem, a quadratic expansion of the search space with increasing database size. Here, a peptide-focused approach is proposed, for which cross-linking peptide candidates are identified in a parallel experiment by using a thiol-cleavable cross-linker with equal reactivity. Samples are reduced and alkylated, thereby releasing cross-linked peptides with a variable modification on the initially cross-linked residue. Modified peptides are identified by database search and concatenated to a peptide database, which is finally used for the analysis of the sample cross-linked with a non-cleavable cross-linker. This way, the search space is dramatically reduced leading to a higher sensitivity, because there is less chance for random hits to false positive sequences. The approach was benchmarked on cross-linked purified protein complexes (20 S Proteasome, yeast PolII, and TFIIH), and in vivo cross-linked bacteria (Bacillus subtilis, Bacillus cereus) by comparing it to the conventional approach of searching against the protein sequences.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Iwan Parfentev  

LAB HEAD: Henning Urlaub

PROVIDER: PXD016554 | Pride | 2020-03-26

REPOSITORIES: Pride

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Dataset's files

Source:
Action DRS
180711_TF2H_pepDB_rev.fasta Fasta
180723_B_Subtilis.fasta Fasta
180724_B_cereus.fasta Fasta
180801_Bcereus_pepDB_rev.fasta Fasta
181109_20S_proteasome_pepDB_rev.fasta Fasta
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Publications

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

Parfentev Iwan I   Schilbach Sandra S   Cramer Patrick P   Urlaub Henning H  

Journal of proteomics 20200319

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 n<sup>2</sup> 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 thos  ...[more]

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