Improved Identification of Peptides, Modification Sites, and Cross-Link Sites by Target-Enhanced Accurate Inclusion...

Chemical cross-linking of proteins coupled with mass spectrometry provides structural insights by identifying cross-linked peptide pairs, abbreviated as cross-links. Presently, cross-link identification suffers from ambiguity and poor sensitivity because they are typically of lower abundance and consequently of lower MS2 quality than linear peptides present in the same sample. Here, we present target-enhanced accurate inclusion mass screening (TAIMS), a meticulously optimized targeted mass spectrometry method. TAIMS significantly improved the quality of MS2, as indicated by fragment ion coverage (FIC) and other metrics. From data-dependent acquisition (DDA) to TAIMS, high-FIC cross-links increased by 359 or 678 on yeast ribosome or Escherichia coli lysate, respectively, or from about 40% to around 90%. As a result, TAIMS substantially enhanced the accuracy of cross-link site localization and mitigated sensitivity loss in cross-link identification from large database searches. The enhanced identification sensitivity of TAIMS is further evidenced by its capacity to recover genuine cross-link identifications from data that would typically be discarded. On cross-linked E. coli lysate, 10.5% (284/2711) of the inclusion-list entries generated from unidentified cross-link-spectrum matches gained identity through TAIMS. Of these, 230 were linear peptides and 54 were cross-links, including 10 intermolecular cross-links missed entirely by DDA. Additionally, we demonstrate that TAIMS is a general method for the identification of low-abundance, post-translationally modified peptides. On a mouse brain sample, TAIMS increased the number of phosphopeptides identified with an accurate phosphosite assignment by 67%. These findings indicate that TAIMS has broad applicability in proteomics.