Transient and weak protein–protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes EIN/HPr and EIIA^Glc/EIIB^Glc as our model systems, we evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities. We showed previously that DOPA2 (di-ortho-phthalaldehyde with a di-ethylene glycol spacer arm) cross-links proteins 60–120 times faster than DSS (disuccinimidyl suberate). We found that though most of the intermolecular cross-links of either cross-linker are consistent with the encounter complexes (ECs), an ensemble of short-lived binding intermediates, more DOPA2 intermolecular cross-links could be assigned to the stereospecific complex (SC), the final lowest-energy conformational state for the two interacting proteins. Our finding suggests that faster cross-linking captures the SC more effectively and cross-linkers of different reactivities potentially probe protein–protein interaction dynamics across multiple timescales.

High-throughput proteomics based on mass spectrometry (MS) analysis has permeated biomedical science and propelled numerous research projects. pFind 3 is a database search engine for high-speed and in-depth proteomics data analysis. pFind 3 features a swift open search workflow that is adept at uncovering less obvious information such as unexpected modifications or mutations that would have gone unnoticed using a conventional data analysis pipeline. In this protocol, we provide step-by-step instructions to help users mastering various types of data analysis using pFind 3 in conjunction with pParse for data pre-processing and if needed, pQuant for quantitation. This streamlined pParse-pFind-pQuant workflow offers exceptional sensitivity, precision, and speed. It can be easily implemented in any laboratory in need of identifying peptides, proteins, or post-translational modifications, or of quantitation based on ¹⁵N-labeling, SILAC-labeling, or TMT/iTRAQ labeling.

Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography–mass spectrometry (LC–MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples—purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow—from sample preparation to LC–MS and data analysis—is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fast-scanning, high-resolution, and accurate-mass LC–MS system.

Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-µmol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure. We further show that the seemingly incompatible cross-links identified with high confidence arise from alternative modes of protein-protein interactions. By converting the intermolecular cross-links to ambiguous distance restraints, we established a rigid-body simulated annealing refinement protocol to seek the minimum set of conformers collectively satisfying the CXMS data. Hence we demonstrate that CXMS allows the depiction of the ensemble structures of protein complexes and elucidates the interaction dynamics for transient and fleeting complexes.