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.

Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen

“…To probe the oligomerization propensity of various IgG constructs, we primarily made use of native MS (Heck, 2008;Diebolder et al, 2014;Wang et al, 2012). Native MS offered sufficient resolution and sensitivity to monitor the monomer-hexamer equilibrium of hexamerization-enhanced IgGs, in which individual oligomeric states could be clearly separated and identified ( Figure 1A).…”

“…Data analysis was conducted with MassLynx (Waters) and Origin Pro (Origin Lab) software. Relative abundances of oligomeric protein species were determined using a previously described method (Wang et al, 2012). The equilibrium molar ratio of C1q:(IgG) 6 to (IgG) 6 was calculated by dividing the total intensity of C1q:(IgG) 6 signals by the total intensity of (IgG) 6 signals detected from solutions containing 2 mM IgG and 0.5 mM C1q.…”

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

“…To solve this problem, in Step 3, we applied mass selection of a subpopulation of ions with limited mass dispersion. 37,38 When subjecting these ions to tandem MS, we used collision-induced dissociation (CID), which yielded not only charge reduced species 39 useful for the MW calculation but also additional constraints enabling more accurate charge calculations. As illustrated in Figure 2B, CID of the mass-selected (mAb) 1 /(sCD38) 2 i + ions (where “ i ” stands for the unknown charge state) results in ( i – 1)+ and ( i – 2)+ charged species (see Figure S1 for detailed analysis) and release of N-terminal segments of the mAb heavy chain, with MW ranging from 11 to 13 kDa (Figure S2A).…”

Substance P Self-Aggregation Revised: A Chromatographic and Mass Spectrometry Analysis

“…On the other hand, liquid chromatography, especially steric exclusion chromatography, appears to be an obvious ̶ although unemployed in the case of SP ̶ technique for analyzing the size distribution of aggregates. Electrospray ionization mass spectrometry (ESI-MS) is a much less used technique for such a kind of analyses: however, it has recently been used to characterize small protein aggregates, and it has been proposed for their semi-quantitative evaluation [14]. The mass spectrometric fragmentation pattern of protonated substance P ions was described to primarily depend on charge status and fragmentation conditions [15], while the aggregation of lysine-containing model peptides was analyzed by ESI-MS by Banerjee and Mazumdar [16] in solvents of different polarity.…”