High-Throughput\nSingle-Particle Characterization of\nAggregation Pathways and the Effects of Inhibitors for Large (Megadalton)\nProtein Oligomers

Protein aggregation is involved in many human diseases,\nbut characterizing\nthe sizes and shapes of intermediate oligomers (∼10–100\nnm) that are important to the formation of macroscale aggregates like\namyloid fibrils is a significant analytical challenge. Here, charge\ndetection mass spectrometry (CDMS) is used to characterize individual\nconformational states of bovine serum albumin oligomers with up to\n∼225 molecules (15 MDa). Elongated, partially folded, and globular\nconformational families for each oligomer can be readily distinguished\nbased on the extent of charging. The abundances of individual conformers\nvary with changes in the monomer concentration or by adding aggregation\ninhibitors, such as SDS, heparin, or MgCl<sub>2</sub>. These results\nshow the potential of CDMS for investigating intermediate oligomers\nin protein aggregation processes that are important for understanding\naggregate formation and inhibition mechanisms and could accelerate\nformulation buffer development to prevent the aggregation of biotherapeutics.