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

Zhen-Lin Chen,Chao Liu,Ji-Li Yin,Yue-He Ding,Si-Min He,Wen-Jing Zhou,Wen-Feng Zeng,Rui-Xiang Sun,Run-Qian Fang,Jia-Ming Meng,Dan Tan,Hao Chi,Meng-Qiu Dong,Yong Cao,Long Wu,Sheng-Bo Fan

doi:10.1038/s41467-019-11337-z

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.

Highlights

We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides
The idea of CXMS had long existed for structural interpretation of proteins, but its practice had been hindered by the lack of software tools
We show that the proposed four target-decoy approach (TDA)-independent evaluation methods are indispensable for systematic evaluation of CXMS search engines

Summary

Introduction

We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. 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. The n-square problem was tackled by the open search strategy, which considers one cross-linked peptide pair as two single peptides, each bearing a modification of large mass yet unknown composition on linkable residues. This strategy identifies candidates for two single peptides individually and recombine the top scored single peptides into cross-linked pairs based on the known mass of precursor[10,12,13,14,17]. As we proposed earlier, a fragment index was introduced to reduce the number of coarse-scored peptides[30]

Methods

Results

Conclusion