Native mass spectrometry, the method by which noncovalent protein complexes are retained in the gas phase for intact mass analysis, is gaining interest as a method for intact protein characterization. The development of a modified orbital ion trap platform for high-resolution analyses has expanded the role of native mass spectrometry to address the challenges of intact protein characterization. Here, we give an overview of the types of analyses that have recently been accomplished with this new instrumentation, ranging from highly decorated small proteins to large, noncovalent complexes that bind small-molecule ligands. These examples highlight the capabilities of native mass spectrometry and hint at the future role in intact protein characterization.
The importance of protein analysis has expanded as the role that proteins play in disease progression and potential treatment has become increasingly evident. Many of the diseases we seek to treat stem from phenomena such as misregulation of proteins or misfolding of protein structure. As a way to combat these types of diseases, there is a push for the use of proteins as therapeutics, most notably the development of disease-specific antibodies (1,2). As an example, monoclonal antibodies (mAbs) have been developed to target human epidermal growth factor receptor 2 (HER2) and tumor necrosis factor (TNF) to treat cancers, rheumatoid arthritis, and Crohn's disease. These antibodies number in the top-selling therapeutics (3). To understand these diseases and develop protein-based treatments, it is necessary to delve into protein structure and interactions to construct a detailed network.
Advances in technology have aided in the characterization of proteins and protein networks. The determination of protein primary sequence is currently routine in many laboratories, but it is the characterization of protein–protein interactions and post-translational modifications (PTMs) that remains challenging. PTM analysis is especially crucial because cellular signals are often transduced through PTMs, such as phosphorylation, and PTMs can result from drug degradation (for example, oxidation). Also, for therapeutic antibodies, glycosylation plays a key role in drug clearance and potential immunogenicity. There are several analytical techniques for studying protein interactions and PTMs, such as ion-exchange chromatography (IEC), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE), and native polyacrylamide gel electrophoresis (PAGE), but mass spectrometry (MS) proteomics methods have become the workhorse for many of these analyses. Often, these interactions and PTMs are detected using enzymatic digestion of a cell lysis or pull-down followed by separation and characterization of the peptides (4). However, this process does not reveal the nature of the intact complexes, and thus these interactions can be hypothesized but not confirmed. Analysis of intact proteins and protein complexes would not only confirm the identity of cellular protein complexes but also provide insight to the multiple proteoforms present (5).MS has become a crucial technology for the characterization of proteins and protein complexes as advances make it more sensitive and robust. MS was initially relegated to the realm of small-molecule analysis because of the inability to transfer proteins to the gas phase efficiently and effectively. Development of "soft" ionization techniques such as electrospray ionization (ESI) broadened the utility of MS by allowing intact proteins to be detected (6). Though the intact protein was then retained in the gas phase, the protein structure was often lost because of the organic solvents and acids used as the ionization solution. The development of nano-ESI, which uses a smaller orifice and lower ionization voltages, allowed the incorporation of buffers consisting of aqueous volatile salts, such as ammonium acetate, as ionization solutions, so that aspects of protein higher-order structure and noncovalent interactions could be retained in the gas phase. This native MS (7) is applicable for a wide array of protein samples, with complexes up to 18 MDa (8,9). Recent developments in MS instrumentation have led to the incorporation of native MS conditions on an orbital ion trap platform (10). This combination has yielded high-resolution analyses of proteins and protein complexes, with the ability to characterize complex, heterogeneous PTMs on an intact level. Here, we highlight some recent applications probed using this technology. These examples span the range of size currently achievable as well as emphasize the ability to characterize multiple proteoforms simultaneously present in a single sample. These applications demonstrate the utility of native MS to provide thorough characterization of heterogeneous mixtures and a basis for the future role of native MS for intact protein analysis.