Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry

Abstract

Protein overexpression and purification are critical for in vitro structure–function characterization studies. However, some proteins are difficult to express in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein-specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to test multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography–mass spectrometry-based method for direct analysis of target proteins in cell lysates. This approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS). While we illustrate a proof of concept here using relatively straightforward examples, the use of IMAC–OBE–nMS to optimize conditions for large-scale protein production may become invaluable for expediting structural biology and biotherapeutic initiatives.

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.…”

Dissecting the structural heterogeneity of proteins by native mass spectrometry

“…However, upon binding prFMN, Fdc1 exists solely as a dimer, and adopts a more compact structure than its apo form (Beveridge et al, 2016). Although some advanced experiments highlighted in this review still involve significant manual steps and/or long data acquisition times, recent developments have established automated native MS workflows (Busch et al, 2021; Hecht, Obiorah, et al, 2022; Orton et al, 2018; Park, Winton, et al, 2020; Ren et al, 2019; VanAernum et al, 2020; Zhang et al, 2003), some of which are commercialized, for high throughput stoichiometry measurement that will complement existing structural biology tools.…”

Protein–Small Molecule Interactions in Native Mass Spectrometry

“…Moreover, the use of high flow rates in ESI results in the formation of relatively large initial droplets (diameter on the order of several micrometers). , Each droplet that is formed can contain multiple analytes, coeluting compounds, and salts, which can compete with the analyte for charge. Thus, ESI is not particularly tolerant for the direct analysis of complex biological systems, or those in which the analytes are challenging to isolate in high yield, and is not typically used unless coupled with online separation and buffer exchange methods that can reduce sample complexity. , …”

“…Thus, ESI is not particularly tolerant for the direct analysis of complex biological systems, or those in which the analytes are challenging to isolate in high yield, and is not typically used unless coupled with online separation and buffer exchange methods that can reduce sample complexity. 76,77 In 1994, Wilm and Mann, building on the early work of Smith 78 and Caprioli 79 into low flow rate ESI sources, reported the development of nanoelectrospray ionization (nanoESI), which addressed some of the major issues associated with traditional ESI sources. 64,80 Although relying on basically the same physical processes to generate gas-phase ions, nanoESI has distinct properties that provide advantages for the analysis of protein−small molecule interactions in native MS. A typical nanoESI emitter consists of a borosilicate or quartz capillary that is pulled such that the internal diameter of the tip is 1−20 μm.…”