Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis.

Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal. In this strategy, followed by the crosslinking reaction, an amine biotin reagent was used to block the unreacted reactive groups on the crosslinker, and subsequently, the crosslinked proteins were tagged by a cleavable biotin-azide ligand based on click chemistry for enrichment. The following crosslinked protein digestion, purification by streptavidin beads, and release by chemical cleavage of the biotin-azide ligand were sequentially performed. In this case, the amine biotin-blocked unhydrolyzed monolinked peptides with the unbreakable arm remained on the streptavidin beads, which realized selective removal without any additional steps. By combining in vivo crosslinking with our proposed MoTE strategy for protein complex analysis of the HeLa cell, the number of high reliability (score 更多