Improved identification of peptides, modification sites, and cross-link sites by Target-enhanced Accurate Inclusion Mass Screening (TAIMS)

ABSTRACT: 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. On cross-linked E. coli lysate or yeast ribosome samples, the percentage of high-FIC cross-links increased from 40.6%~43.3% by data-dependent acquisition (DDA) to 87.0%~94.5% by TAIMS. 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. 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% of the inclusion-list entries generated from unidentified cross-link-spectrum matches gained identity through TAIMS, of which 65.5% turned out to be linear peptides and 19.0% being cross-links, including 10 inter-molecular cross-links missed entirely by DDA. Overall, TAIMS increased high-confidence cross-link identifications by 52.9%~131.3%. Additionally, we demonstrate that TAIMS is a general method for identification of low-abundance, post-translationally modified peptides. On a mouse brain sample, TAIMS increased the number of phosphopeptides identified with accurate phosphosite assignment by 67%. These findings indicate that TAIMS holds broad applicability in proteomics.

ORGANISM(S): Escherichia Coli Mus Musculus Artificial Sequences Bos Taurus Saccharomyces Cerevisiae

SUBMITTER: Mengqiu Dong  

PROVIDER: PXD065604 | iProX | Sun Jun 28 00:00:00 BST 2026

REPOSITORIES: iProX