A Novel Sec-ms Method for the Studyof Proteins and Their Interactions

Size exclusion chromatography (SEC) is widely used for molecular weight estimations of proteins in their native state. SEC has found applications in studies on protein purity, protein-protein interactions and protein aggregation. The major limitations of SEC are that the retention time of proteins can be affected by their hydrodynamic properties and also due to tendency of some proteins to interact with the column matrix. A hyphenated size exclusion-mass spectrometric method (SECMS) was developed for the study of proteins and their interactions. The method utilizes a mass spectrometry compatible mobile phase consisting of ammonium formate with a post-column addition of acetonitrile and formic acid. SEC-MS combines the high mass accuracy of electrospray ionization mass spectrometry with the ability of SEC to resolve protein complexes. We found that noncovalent protein complexes do not survive electrospray ionization and overlapping isotopic envelopes are obtained for monomeric, dimeric, and aggregates of BSA, which after deconvolution also yield identical molecular weights for the three isoforms. This property of identifying monomeric molecular weights of proteins in a multimeric complex is unique to SEC-MS. Other methods such as on-line light-scattering, absorbance, and refractive index detectors can only measure the mass of intact complex and hence, cannot identify different components in a protein complex. We have shown that this novel SEC-MS method can be very effective in the study of covalent and noncovalent protein aggregation, and in the analysis of IgG1. EXPERIMENTAL –