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 30-800kDa. 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.
Highlights
Over the last two decades, native mass spectrometry has become a powerful analytical method to analyze large proteins and protein complexes [1,2,3]
A standard protein mix composed of carbonic anhydrase II (CA, 29 kDa), alcohol dehydrogenase (ADH, kDa), NIST monoclonal antibody (NIST mAb, kDa), and pyruvate kinase (PK, 231 kDa) was utilized for nCZE-TDMS method development
MS1 parameters were optimized via direct infusion through the CESI system for each standard individually
Summary
Over the last two decades, native mass spectrometry (nMS) has become a powerful analytical method to analyze large proteins and protein complexes [1,2,3]. Aqueous buffer systems close to neutral pH are used to preserve endogenous (“native”) characteristics of proteins and their non-covalent interactions [4,5] In this way, the composition of biomolecular structures can be studied in detail, leading to a better understanding of their topology, stoichiometry, and biological functions [6,7]. Fewer protonation events take place during native electrospray ionization, and lower and fewer charge states for proteins are observed [9] This characteristic can lead to increased sensitivity, especially for large proteins and their complexes. Various fragmentation techniques and their combination improved the capabilities of subunit ejection and subsequent fragmentation in nTDMS [22,23,24,25,26] Despite all these advancements in technology, ion suppression and signal superposition cannot be completely avoided in a direct infusion nMS experiment, especially for complex samples. Upfront separation under native conditions is beneficial and highly desired
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