Protein-Protein Interaction Network Mapping by Affinity Purification Cross-Linking Mass Spectrometry (AP-XL-MS) based Proteomics.

Protein-protein interactions (PPIs) are the physical interactions formed among proteins. These interactions are primarily functional, i.e., they arise from specific biomolecular events, and each interaction interface serves a specific purpose. A significant number of methods have been developed for protein interactions in the field of proteomics in the last decade. Advanced mass spectrometry technology significantly contributed to the development of these methods. The rapid advancement of groundbreaking MS technology has greatly aided the mapping of protein interaction from large-data sets comprehensively. This chapter describes the affinity purification (AP) mass spectrometry (MS)-based methods combined with chemical cross-linking (XL) of protein complexes. This chapter includes sample preparation methods involving cell culture, cell treatments with ligands, drugs, and cross-linkers, protein extractions, affinity purification, sodium dodecyl sulfate (SDS) polyacrylamide gel separation, in-solution or in-gel digestion, liquid-chromatography, and mass spectrometry analysis of samples (LC-MS/MS). Application of a cleavable cross-linker, dual cleavable cross-linking technology (DUCCT) in combination with the affinity purification (AP) method has also been described. Methods for data analysis using unmodified and cross-linked peptide analysis are discussed.