Nonspecific cross-linker can provide distance restraints between surface residues of any type, which could be used to investigate protein structure construction and protein–protein interaction (PPI). However, the vast number of potential combinations of cross-linked residues or sites obtained with such a cross-linker makes the data challenging to analyze, especially for the proteome-wide applications. Here, we developed SpotLink software for identifying site nonspecific cross-links at the proteome scale. Contributed by the dual pointer dynamic pruning algorithm and the quality control of cross-linking sites, SpotLink identified > 3000 cross-links from human cell samples within a short period of days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on the datasets of the simulated succinimidyl 4,4′-azipentanoate dataset and the condensin complexes with known structures. In addition, some valuable PPI were discovered in the datasets of the condensin complexes and the HeLa dataset, indicating the unique identification advantages of site nonspecific cross-linking. These findings reinforce the importance of SpotLink as a fundamental characteristic of site nonspecific cross-linking technologies.

Data-dependent liquid chromatography–tandem mass spectrometry (LC–MS/MS) is widely used in proteomic analyses. A well-performed LC–MS/MS workflow, which involves multiple procedures and interdependent metrics, is a prerequisite for deep proteome profiling. Researchers have previously evaluated LC–MS/MS performance mainly based on the number of identified peptides and proteins. However, this is not a comprehensive approach. This motivates us to develop MSRefine, which aims to evaluate and optimize the performance of the LC–MS/MS workflow for data-dependent acquisition (DDA) proteomics. It extracts 47 kinds of metrics, scores the metrics, and reports visual results, assisting users in evaluating the workflow, locating problems, and providing optimizing strategies. In this study, we compared and analyzed multiple pairs of datasets spanning different samples, methods, and instruments and demonstrated that the comprehensive visual metrics and scores in MSRefine enable us to evaluate the performance of the various experiments and provide optimal strategies for the identification of more peptides and proteins.

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Migraine is a neurovascular disease with a high disability rate. Acupuncture treatment has emerged as a safe and viable alternative prophylactic therapy that can effectively alleviate the duration and frequency of migraine attacks. However, the therapeutic mechanisms underlying the effects of acupuncture are yet to be systematically elucidated. In this study, we enrolled female patients with migraine without aura (n = 20) and healthy controls (n = 10). Patients received acupuncture treatment on DU20, DU24, bilateral GB13, GB8, and GB20, applied three times per week over the course of 4 weeks for 12 sessions in total. Blood samples were collected from the median cubital vein before and after acupuncture treatment. Proteomic and metabolomic profiling was performed using liquid chromatography-mass spectrometry to determine the characteristics of differentially expressed molecules and expression of their corresponding biological pathways as well as to elucidate the pathogenesis of migraine and the biological effects underlying the treatment of migraine with acupuncture. Proteomic and metabolomic profiling of plasma samples from patients with migraine without aura before and after acupuncture treatment revealed enrichment of immune-related pathway functions and the arginine synthesis pathway. Joint pathway analyses revealed significant enrichment of the pentose phosphate and glycolysis/gluconeogenesis pathways in patients with migraine. The glycolysis/gluconeogenesis and riboflavin metabolism pathways were significantly enriched after acupuncture treatment. The expression levels of various key proteins and metabolites, including α-D-glucose, flavin adenine dinucleotide, biliverdin reductase B, and L-glutamate, were significantly differentially expressed before and after acupuncture treatment in patients with migraine without aura. Treatment of migraine with acupuncture was associated with significant changes in key molecules and pathways, indicative of physiological changes in the trigeminovascular system, glutamate neurotoxicity, and other migraine-related physiological changes. Overall, our comprehensive analysis using proteomic and metabolomic profiling demonstrates that energy metabolism may serve as a key correlative link in the occurrence of migraine and the therapeutic effects of acupuncture treatment. Our findings may facilitate the identification of diagnostic and therapeutic modalities in the ongoing search for effective treatments for migraine attacks.