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, 15 N 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.

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO. The R-R or K-R cross-links generated by ArGO or KArGO fit well with protein crystal structures and provide information not attainable by K-K cross-links. KArGO, in particular, is highly valuable for CXMS, with robust performance on a variety of samples including a kinase and two multi-protein complexes. In the case of the CNGP complex, KArGO cross-links covered as much of the PPI interface as R-R and K-K cross-links combined and improved the accuracy of Rosetta docking substantially.

The widely adopted the high strength steel tube is an important way to solve the lightweight body and collision safety. The traditional high pressure tube hydroforming and hot gas forming through “expansion” deformation to form hollow parts, and the internal pressure is the deformation driving force. There are some industrial difficulties such as heavy dependence on high pressure, wall thickness thinning and low production efficiency, which limits the production and application of high-strength steel tubes. Based on the principle of compression deformation, this paper develops the hot hydro-forging process to form the high-strength steel tubes. After the tube blank is heated to austenitization, the tube is compressed and deformed by the die closing force of the die through a new die structure. The internal pressure only plays a supporting role, which fundamentally avoids the thickness thinning and cracking under the expansion deformation mode, and has the advantages of rapid forming time and uniform quenching. Finally, the A-pillar tube with 1500 MPa is formed through the reasonable hot hydro-forging process.