title: Characterization of small protein aggregates and oligomers using size exclusion chromatography with online detection by native electrospray ionization mass spectrometry.
abstract: Self-association of proteins is important in a variety of processes ranging from acquisition of native quaternary structure (where the association is tightly controlled and proceeds in a highly ordered fashion) to aggregation and amyloidosis. The latter is frequently accompanied (or indeed triggered) by the loss of the native structure, but a clear understanding of the complex relationship between conformational changes and protein self-association/aggregation remains elusive due to the great difficulty in characterizing these complex and frequently heterogeneous species. In this study, size exclusion chromatography (SEC) was used in combination with online detection by native electrospray ionization mass spectrometry (ESI MS) to characterize a commercial protein sample (serum albumin) that forms small aggregates. Although noncovalent dimers and trimers of this protein are readily detected by native ESI MS alone, combination of SEC and ESI MS allows a distinction to be made between the oligomers present in solution and those formed during the ESI process (artifacts of ESI MS). Additionally, native ESI MS detection allows a partial loss of conformation integrity to be detected across all albumin species present in solution. Finally, ESI MS detection allows these analyses to be carried out readily even in the presence of other abundant proteins coeluting with albumin. Native ESI MS as an online detection method for SEC also enables meaningful characterization of species representing different quaternary organization of a recombinant glycoprotein human arylsulfatase A even when their rapid interconversion prevents their separation on the SEC time scale.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Khaja
authors.initials: K
authors.lastname: Muneeruddin
authors.forename: John J
authors.initials: JJ
authors.lastname: Thomas
authors.forename: Paul A
authors.initials: PA
authors.lastname: Salinas
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D001704:Biopolymers / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D001704:Biopolymers; D011506:Proteins