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

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2021 Jun 8

PMID 34100589

Citations 10

Authors

Florian Busch

Zachary L VanAernum

Stella M Lai

Venkat Gopalan

Vicki H Wysocki

Affiliations

Soon will be listed here.

Abstract

Protein overexpression and purification are critical for 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.

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