Standard procedures for native CZE‐MS of proteins and protein complexes up to 800 kDa

Abstract

Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving "native" non-covalent inter-and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high-quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30-800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE-MS setup. This study is aimed to help normalize nCZE-MS practices to enhance the CE community and provide a resource for the production of reproducible and high-quality data.

Novel Strategies to Address the Challenges in Top-Down Proteomics

“…There is also significant interest in the development of capillary electrophoresis-MS (CE-MS) as a non-denaturing separation technique. − The wide variety of orthogonality of separation selectivity to conventional LC-MS methods and low sample volume requirements have made CE-MS a very attractive technique for native separations. , As new commercial systems and discrete devices are developed, , we anticipate that CE-MS will rise to increased prominence in top-down proteomics.…”

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

“…Native mass spectrometry (nMS) has emerged as a powerful analytical tool for the delineation of protein complexes. − Due to the heterogeneous nature of protein complexes, coupling online and native liquid-phase separations to nMS is an ideal approach for protein complex analysis. Various liquid-phase separation techniques, such as size exclusion chromatography, , ion-exchange chromatography, and capillary zone electrophoresis (CZE), − have been coupled directly to nMS for the extensive characterization of protein complexes in simple to complex samples. However, the separation resolution of the liquid chromatography (LC) and CZE techniques for protein complexes still need to be improved.…”

Approaches to Heterogeneity in Native Mass Spectrometry

“…For example, FT/GT methods can greatly facilitate identification of charge states for distinct, highly overlapped peaks in mass spectra, thus inputting the range of charge states thereby identified may greatly reduce artifacts of other deconvolution methods that perform best when the charge state range is confined to correct values. Experimental and instrumental improvements possible in the near future include development of robust inlet-based separation beyond liquid chromatography (such as capillary zone electrophoresis − ), next-generation nESI tip design (including reliable production of submicron emitters ,,,,,, and theta-glass emitters for rapid mixing of samples during the ESI process − ), and more efficient in-source desolvation. , …”

“…In these sections we draw upon the numerous comprehensive reviews of these specific topics. Although many online solution-phase separation approaches (such as online chromatography methods − and capillary zone electrophoresis − ) have been introduced to help solve this problem, our discussion is confined to instrumentation and techniques commonly available within mass spectrometers themselves or with small modifications. As this review is written from an academic viewpoint, we refer readers not only to recent native MS work on biotherapeutics ,− but also to many recent efforts and helpful perspectives on this topic from industry scientists representing a variety of biopharmaceutical companies, drawing upon these insights where possible. ,,,− Importantly, though implementation of the methods we describe below faces unique challenges in industry (namely, rigorous standardization and commercialization), we hold that educating potential users on the theoretical aspects of these approaches is important and beneficial to all who utilize native MS, regardless of background.…”