Native CZE-TDMS for the characterization of proteins and protein complexes up to 800 kDa

Native mass spectrometry (nMS) is a rapidly growing field for the characterization of large proteins and protein complexes, preserving endogenous (“native”) non-covalent interactions. So far, direct infusion of purified proteins into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance step while consuming trace amounts of sample material. Furthermore, Top-down mass spectrometry (TDMS) offers complete molecular information (i.e., in-depth characterization of proteoforms). Therefore, native CZE in combination with TDMS represents a promising concept to face and overcome current and future challenges in the field of proteomics.
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. A standard protein mix composed of carbonic anhydrase II (29 kDa), alcohol dehydrogenase (147 kDa), NIST monoclonal antibody (148 kDa), and pyruvate kinase (231 kDa) was used as a model system. The mixture covers a wide range of targets including protein-metal complexes, protein-protein complexes, and biopharmaceuticals. The influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, ejection/fragmentation parameters were optimized for the native top-down analysis of intact complexes, expected to also be applicable to other CE-MS interfaces systems (e.g. sheath liquid systems). As a showcase for high-molecular weight species (>250 kDa), we selected the protein complex GroEL (~800 kDa), a chaperonin 60 tetradecamer. The intact GroEL complex was detected by nCZE-TMS and its major proteoforms subunits could be identified after ejection followed by HCD fragmentation. Additionally, the developed system was later applied for the separation and characterization of proteoforms derived from endogenous nucleosomes.
Based on the high-performance separation capability added to nMS, we expect a wide range of potential applications for nCZE-TDMS in proteomics, including fields such as structural biology and biomedicine.