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A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides.

ABSTRACT: We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. With a two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, using simulated datasets, synthetic datasets, ¹⁵N metabolically labeled datasets, and entrapment databases, four analysis methods were designed to evaluate the credibility of ten state-of-the-art search engines. This systematic evaluation shows that pLink 2 outperforms these methods in precision and sensitivity, especially at proteome scales. Lastly, re-analysis of four published proteome-scale cross-linking datasets with pLink 2 required only a fraction of the time used by pLink 1, with up to 27% more cross-linked residue pairs identified. pLink 2 is therefore an efficient and reliable tool for cross-linking mass spectrometry analysis, and the systematic evaluation methods described here will be useful for future software development.

SUBMITTER: Chen ZL

PROVIDER: S-EPMC6667459 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides.

Chen Zhen-Lin ZL Meng Jia-Ming JM Cao Yong Y Yin Ji-Li JL Fang Run-Qian RQ Fan Sheng-Bo SB Liu Chao C Zeng Wen-Feng WF Ding Yue-He YH Tan Dan D Wu Long L Zhou Wen-Jing WJ Chi Hao H Sun Rui-Xiang RX Dong Meng-Qiu MQ He Si-Min SM

Nature communications 20190730 1

We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. With a two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, using simulated datasets, synthetic datasets, <sup>15</sup>N metabolically labeled datasets, and entrapment databases, four analysis methods were designed to evaluate the credibility of ten state-of-the-art se ...[more]

PMID: 31363125

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Project description:Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes. We report herein the development of a user-friendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect cross-linked peptides from the digests of large (>or=170-kDa) cross-linked proteins, i.e. conjugates. Our strategy combines the use of a low excess of cross-linker, data base searching, and Fourier transform ion cyclotron resonance MS to experimentally minimize and theoretically cull the side products of cross-linking. Using this strategy, the (alpha beta gamma delta)(4) phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa betagamma conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa beta subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the beta subunit and suggests a dynamic structural role for the regulatory N terminus of that subunit. The results offer proof of concept for the CrossSearch strategy for analyzing conjugates and are the first to reveal a tertiary structural element of either homologous alpha or beta regulatory subunit of phosphorylase kinase.

Project description:BackgroundChemical cross-linking combined with mass spectrometry (CX-MS) is a high-throughput approach to studying protein-protein interactions. The number of peptide-peptide combinations grows quadratically with respect to the number of proteins, resulting in a high computational complexity. Widely used methods including xQuest (Rinner et al., Nat Methods 5(4):315-8, 2008; Walzthoeni et al., Nat Methods 9(9):901-3, 2012), pLink (Yang et al., Nat Methods 9(9):904-6, 2012), ProteinProspector (Chu et al., Mol Cell Proteomics 9:25-31, 2010; Trnka et al., 13(2):420-34, 2014) and Kojak (Hoopmann et al., J Proteome Res 14(5):2190-198, 2015) avoid searching all peptide-peptide combinations by pre-selecting peptides with heuristic approaches. However, pre-selection procedures may cause missing findings. The most intuitive approach is searching all possible candidates. A tool that can exhaustively search a whole database without any heuristic pre-selection procedure is therefore desirable.ResultsWe have developed a cross-linked peptides identification tool named ECL. It can exhaustively search a whole database in a reasonable period of time without any heuristic pre-selection procedure. Tests showed that searching a database containing 5200 proteins took 7 h. ECL identified more non-redundant cross-linked peptides than xQuest, pLink, and ProteinProspector. Experiments showed that about 30 % of these additional identified peptides were not pre-selected by Kojak. We used protein crystal structures from the protein data bank to check the intra-protein cross-linked peptides. Most of the distances between cross-linking sites were smaller than 30 Å.ConclusionsTo the best of our knowledge, ECL is the first tool that can exhaustively search all candidates in cross-linked peptides identification. The experiments showed that ECL could identify more peptides than xQuest, pLink, and ProteinProspector. A further analysis indicated that some of the additional identified results were thanks to the exhaustive search.

Dataset Information

A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides.

Publications

A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides.

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