The S/T/Y-linked residue pairs identified by current software are unreliable
Ontology highlight
ABSTRACT:
ORGANISM(S): Bos Taurus
SUBMITTER: 
Mengqiu Dong
PROVIDER: PXD039103 | iProX | Mon Dec 26 00:00:00 GMT 2022
REPOSITORIES: iProX
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Project description:Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel MS-cleavable reagents. These cross-linkers enhance the reliability of cross-link identification. Presently, only water-insoluble MS-cleavable cross-linkers are commercially available. However, the comprehensive analysis of the various chemical and structural motifs inherent in proteins depends on the targeting of different protein sites with varying degrees of hydrophilicity. We introduce a new MS-cleavable cross-linker called disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group. It therefore serves as a water-soluble counterpart to the commercially available and widely used disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers—bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU on bovine serum albumin. We also compared efficacy of DSBU and DSSBU on human hemoglobin. Our results demonstrate that the superior water solubility of DSSBU enables to avoid the usage of polar solvents such as DMSO but still maintaining the effectivity of MS-cleavable crosslinker such as DSBU.
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Project description: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.
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