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Mass spectrometry of intact proteins and protein complexes has the potential to provide a transformative level of information on biological systems, ranging from sequence and post-translational modification analysis to the structures of whole protein assemblies. This ambitious goal requires the efficient fragmentation of both intact proteins and the macromolecular, multicomponent machines they collaborate to create through noncovalent interactions. Improving technologies in an effort to achieve such fragmentation remains perhaps the greatest challenge facing current efforts to comprehensively analyze cellular protein composition and is essential to realizing the full potential of proteomics. In this work, we describe the use of a trimethyl pyrylium (TMP) fixed-charge covalent labeling strategy aimed at enhancing fragmentation for challenging intact proteins and intact protein complexes. Combining analysis of TMP-modified and unmodified protein complexes results in a greater diversity of regions within the protein that give rise to fragments, and results in an up to 2.5-fold increase in sequence coverage when compared to unmodified protein alone, for protein complexes up to 148 kDa. TMP modification offers a simple and powerful platform to expand the capabilities of existing mass spectrometric instrumentation for the complete characterization of intact protein assemblies.

The pharmaceutical industry continues to expand its search for innovative biotherapeutics. The comprehensive characterization of such therapeutics requires many analytical techniques to fully evaluate critical quality attributes, making analysis a bottleneck in discovery and development timelines. While thorough characterization is crucial for ensuring the safety and efficacy of biotherapeutics, there is a need to further streamline analytical characterization and expedite the overall timeline from discovery to market. This review focuses on recent developments in liquid-phase separations coupled with ion mobility-mass spectrometry (IM-MS) for the development and characterization of biotherapeutics. We cover uses of IM-MS to improve the characterization of monoclonal antibodies, antibody-drug conjugates, host cell proteins, glycans, and nucleic acids. This discussion is based on an extensive literature search using Web of Science, Google Scholar, and SciFinder. IM-MS has the potential to enhance the depth and efficiency of biotherapeutic characterization by providing additional insights into conformational changes, post-translational modifications, and impurity profiles. The rapid timescale of IM-MS positions it well to enhance the information content of existing assays through its facile integration with standard liquid-phase separation techniques that are commonly used for biopharmaceutical analysis. [ABSTRACT FROM AUTHOR]

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer's disease.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98–CBM48–GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut. [ABSTRACT FROM AUTHOR]