MaXLinker: proteome-wide cross-link identifications with high specificity and sensitivity

Abstract

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry has the unique capability to detect proteinprotein interactions at a large scale along with spatial constraints between interaction partners.However, the current cross-link search algorithms follow an "MS2-centric" approach and, as a result, suffer from a high rate of mis-identified cross-links (~15%). We address this urgent problem, by designing a novel "MS3-centric" approach for cross-link identification and implemented it as a search engine called MaXLinker. MaXLinker significantly outperforms the current state of the art search engine with up to 18-fold lower false positive rate. Additionally, MaXLinker results in up to 31% more cross-links, demonstrating its superior sensitivity and specificity. Moreover, we performed proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we unveiled the most comprehensive set of 9,319 unique cross-links at 1% false discovery rate, comprising 8,051 intraprotein and 1,268 interprotein cross-links.Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

Towards a structurally resolved human protein interaction network

“…These interactions were further enriched by obtaining annotations on cross-linking peptides matched across pairs of interactions, disease related mutations and protein phosphorylation sites in the selected proteins. Crosslink data was collected from (Yugandhar et al, 2020;Schweppe et al, 2016;Klykov et al, 2020;Steigenberger et al, 2019;Klykov et al, 2018;Fasci et al, 2018;Eliseev et al, 2018;Gestaut et al, 2019;Klatt et al, 2020;Sabath et al, 2020;Mohamed et al, 2021), filtered for peptides assigned to only one sequence. Clinical missense variants associated with disease were collected from ClinVar.…”

Structure and mechanism of TRAPPIII-mediated Rab1 activation

“…For MS3, ions were fragmented in the linear ion trap using higher-energy collisional dissociation (HCD) at 35% HCD energy. The cross-link search was performed using MaXLinker software (Yugandhar et al, 2020). The structure mapping of cross-links was performed using Xlink Analyzer (Kosinski et al, 2015) implemented in UCSF Chimera (Pettersen et al, 2004).…”

Progress in methodologies and quality‐control strategies in protein cross‐linking mass spectrometry

“…Similarly, the large‐scale and proteome‐wide XL‐MS studies tend to identify much higher fraction of cross‐links from highly abundant complexes such as ribosome and proteasome compared to that of proteins with relatively lower abundance [20]. To examine this comprehensively, we compiled a set of 31,745 cross‐links among 4035 proteins from published datasets [21–38] and analyzed them in relation to the cellular abundance of the cross‐linked proteins, obtained from PaxDB [39] (3688 out 4035 proteins had abundance values in the PaxDB which contribute to 28,244 cross‐links in the dataset) (Figure 2A, 2B). The results showed that about 76% of the cross‐links map to the proteins that belong to the top 10% category in terms of their cellular abundance (Figure 2B).…”