Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

Abstract

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes

“…This is advantageous for protein complexes that are difficult to express or purify and only available in small quantities. An alternative technique that can be used for the desalting of protein complexes is rapid online buffer exchange, which has shown the ability to remove nonvolatiles from different buffer conditions . However, submicrometer capillaries can be used when HPLC equipment is unavailable and if high throughput is not needed.…”

Oxidized and Reduced Dimeric Protein Complexes Illustrate Contrasting CID and SID Charge Partitioning

“…During heating, the unfolding of the monomer causes loss of the helical structure and allows the Cys121 to reversibly create a Cys106–Cys121 disulfide and a free thiol at Cys 119, which induces the aggregation . In order to maintain the interface of the BLG dimer and minimize overall structural change, we optimized the reduction conditions by monitoring the mass shift of the proteins with online buffer exchange native MS. , We found that in 50 mM dithiothreitol, after 20 min incubation at 50 °C, the average mass increased by 2 Da for each subunit after reduction (Figure S2) which indicates the reduction of only one disulfide bond. Presumably only the water-accessible disulfide bond Cys66–Cys160 has undergone reduction.…”

“…Accurate masses were measured on an Exactive Plus EMR Orbitrap instrument (Thermo Scientific) modified with a quadrupole mass filter and a surface-induced dissociation device . The samples were injected on a self-packed buffer exchange column (Bio-Rad P 6̅ packing material) using an Ultimate 3000 RSLC (Thermo Scientific) coupled to the modified Exactive Plus EMR Orbitrap instrument (Thermo Scientific). , The mobile phase was 200 mM ammonium acetate. The flow rate was 100 μL/min, and proteins typically eluted within 1.2 min.…”