We describe optimized fragmentation schemes and data analysis strategies substantially enhancing the depth and accuracy in identifying protein cross-links using non-restricted whole proteome databases. These include a novel hybrid data acquisition strategy to sequence cross-links at both MS2 and MS3 level and a new algorithmic design XlinkX v2.0 for data analysis. As proof-of-concept we investigated proteome-wide protein interactions in E. coli and HeLa cell lysates, respectively, identifying 1,158 and 3,301 unique cross-links at ∼1% false discovery rate. These protein interaction repositories provide meaningful structural information on many endogenous macromolecular assemblies, as we showcase on several protein complexes involved in translation, protein folding and carbohydrate metabolism.

Mitochondria exert an immense amount of cytophysiological functions, but the structural basis of most of these processes is still poorly understood. Here we use cross-linking mass spectrometry to probe the organization of proteins in native mouse heart mitochondria. Our approach provides the largest survey of mitochondrial protein interactions reported so far. In total, we identify 3,322 unique residue-to-residue contacts involving half of the mitochondrial proteome detected by bottom-up proteomics. The obtained mitochondrial protein interactome gives insights in the architecture and submitochondrial localization of defined protein assemblies, and reveals the mitochondrial localization of four proteins not yet included in the MitoCarta database. As one of the highlights, we show that the oxidative phosphorylation complexes I-V exist in close spatial proximity, providing direct evidence for supercomplex assembly in intact mitochondria. The specificity of these contacts is demonstrated by comparative analysis of mitochondria after high salt treatment, which disrupts the native supercomplexes and substantially changes the mitochondrial interactome.

The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements not only benefitted MS‐based high‐throughput proteomics but also increased the impact of mass spectrometry on the field of structural and molecular biology. Here, we review how state‐of‐the‐art MS methods, including native MS, top‐down protein sequencing, cross‐linking‐MS, and hydrogen–deuterium exchange‐MS, nowadays enable the characterization of biomolecular structures, functions, and interactions. In particular, we focus on the role of mass spectrometry in integrated structural and molecular biology investigations of biological macromolecular complexes and cellular machineries, highlighting work on CRISPR–Cas systems and eukaryotic transcription complexes.