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Screening_tagged_proteins_using_tandem_affinity-buffer_exchange_chromatography_online_with_native_mass_spectrometry-1.pdf (3.76 MB)

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

submitted on 15.02.2021, 15:58 and posted on 16.02.2021, 13:23 by Florian Busch, Zachary VanAernum, Stella M. Lai, Venkat Gopalan, Vicki Wysocki
Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express robustly 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 screen
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 rapid, direct analysis of target proteins in cell lysates. This online approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based buffer exchange (OBE) and native mass spectrometry (nMS). The use of IMAC-OBE-nMS to optimize conditions for large-scale protein production should expedite structural biology and biotherapeutic initiatives.


Resource for Native Mass Spectrometry Guided Structural Biology

National Institute of General Medical Sciences

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Email Address of Submitting Author


The Ohio State University


United States of America

ORCID For Submitting Author


Declaration of Conflict of Interest


Version Notes

Version 1.0