There are three levels of protein interactions: multi-subunit proteins, multi-component protein complexes, and transient, ordered associations between different proteins. In particular, the last protein interaction exists widely in living organisms and plays a role in various life activities such as material metabolism, signal transduction, cell growth and death. Commonly used methods to study protein interactions are: tandem affinity purification, co-immunoprecipitation, yeast two hybrid, phage display and chemical cross-linking, etc.
Among them, chemical cross-linking of proteins coupled with mass spectrometry (XL-MS)technique captures the protein complexes of interest that have interacted with each other and combines with the high resolution capability of biological mass spectrometry to obtain the advanced structural information of proteins in a rapid and high-throughput manner, which gradually become a promising method for studying protein interactions. Therefore, XL-MS has been widely used in the past few years, often aided by electron microscopy and structural modeling, and has become a powerful tool for resolving the three-dimensional structure of large protein complexes.
Identification of cross-linked residues can provide a map of interacting protein surfaces that can be used for:
1. Building direct PPI networks (versus inferred);
2. Mapping PPI interfaces for integrative structural determination of protein complexes.
The significant advantage of this method is that it can "capture" weak, transient interactions in a specific state.
In addition, in vivo analysis can also be performed if a membrane-permeable cross-linking agent is used.
Compared with traditional methods, XL-MS has low requirements on sample volume and sample purity, rapid analysis and high throughput.
Creative Proteomics utilizes XL-MS methods to analyze the pathways and structures of hard-to-characterize protein complexes in vitro and in vivo. These complexes include transiently interacting complexes (eg, substrate-enzyme complexes), flexible subunits, heterogeneous compositions or conformations, and subunits with poor solubility or stability.
The method can be divided into four steps: cross-linking reaction of the sample, cross-linking product purification, mass spectrometry analysis, and data processing.
1. The first step is to select a suitable chemical cross-linking agent according to the purpose of the study and perform a chemical cross-linking reaction in the biological sample to produce the target cross-linking product.
2. Due to the complexity of protein molecules, the diversity of protein components in biological samples and the relative specificity of chemical cross-linking agents, as well as the pH and ionic strength of the microenvironment during the reaction, the complexity of the reaction is greatly increased, and the target cross-linking product is only a very limited part of it. Therefore, purification of the target cross-linked product is a very important step, which largely determines the success or failure of the whole study.
3. The target cross-linked product is digested with protease to make it a peptide that can be detected by mass spectrometry.
4. This is followed by liquid chromatography (LC) separation and identification by mass spectrometry (MS) analysis. This workflow enables the study of protein-protein interactions by maintaining the original interacting complexes.
Typical XL-MS workflow