High-Mass Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Absolute Quantitation of Noncovalent Protein–Protein Binding Interactions

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging MALDI-MS. Herein, we successfully used commercially available purified proteins (?-galactosidase BSA) as internal standards MALDI-MS analysis achieved absolute quantification several analytes. We systematically evaluated four sample deposition methods, using the sandwich method with saturated sinapinic acid top layer, performed by Combined chemical cross-linking, this strategy was further evaluate affinity protein-protein interactions (PPIs), specifically two soluble protein receptors (interleukin 1 receptor interleukin 2 receptor) membrane (rhodopsin angiotensin 1) interaction partners. The measured dissociation constants complexes formed were between 10 nM 5 ?M. expect high-throughput, rapid method, which does not require labeling or immobilization any partners, become viable alternative traditional biophysical methods PPIs.