Evaluation of Electrospray Ionization Mass Spectrometry as a Tool for Characterization of Small Soluble Protein Aggregates

Abstract

Protein aggregation continues to attract significant interest in many areas of biology and medicine not only due to its pivotal role in the etiology of conformational diseases (such as Parkinson's and Alzheimer's) but also due to its importance in the biopharmaceutical sector, where aggregation of protein therapeutics exerts a deleterious effect on their efficacy and safety. Despite the tremendous success of electrospray ionization mass spectrometry (ESI MS) in a large number of studies of noncovalent protein interactions, application of this technique to study aggregation processes has been very limited so far, and lower resolution techniques, such as size exclusion chromatography (SEC) and analytical ultracentrifugation, remain the default tools in characterizing small soluble protein aggregates. In this work we used heat-stressed human antithrombin III (AT), a 58 kDa glycoprotein, to compare SEC and ESI MS as a means to probe composition of the complex mixture of soluble oligomeric species generated by heat-induced aggregation. SEC allows several oligomeric species to be observed and collected, followed by their identification with ESI MS. The same oligomeric species can be also directly observed in the ESI MS of the unfractionated sample of the heat-stressed AT. The abundance distribution of these small soluble aggregates in ESI MS and SEC cannot be compared directly, since the ESI signal is linked to the molar concentration of the analyte in solution, whereas the UV absorption detection in SEC reports weight concentration. However, once the appropriate corrections are made, the abundance of the small aggregates derived from ESI MS becomes remarkably close to that calculated based on SEC data, suggesting that ESI MS may be directly applied for semiquantitative characterization of soluble protein aggregates.