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

Biochemistry
Florian BuschVicki H Wysocki

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.

References

Jul 1, 1985·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·F ShermanS Tsunasawa
Oct 31, 1988·Biochemical and Biophysical Research Communications·L B TsaiM B Mann
Feb 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·S Tabor, C C Richardson
Dec 3, 1998·Journal of the American Society for Mass Spectrometry·L Konermann, D J Douglas
Dec 21, 2002·Methods in Molecular Biology·Jeffrey D Fox, David S Waugh
Jun 21, 2003·Clinical Chemistry·Thomas M Annesley
Jun 16, 2004·Cellular and Molecular Life Sciences : CMLS·C GiglioneT Meinnel
Jun 26, 2004·Advances in Biochemical Engineering/biotechnology·Aleksei Rozkov, Sven-Olof Enfors
Jul 21, 2004·Mass Spectrometry Reviews·Albert J R Heck, Robert H H Van Den Heuvel
Oct 27, 2004·Trends in Biochemical Sciences·Neelan J MarianayagamJacqueline M Matthews
Aug 16, 2005·Analytical Chemistry·Igor A Kaltashov, Anirban Mohimen
Jul 4, 2006·Biochimica Et Biophysica Acta·Victor Martin Bolanos-Garcia, Owen Richard Davies
Apr 5, 2007·Nature Protocols·Helena Hernández, Carol V Robinson
Feb 1, 2008·Nature Methods·UNKNOWN Structural Genomics ConsortiumKristin C Gunsalus
Feb 12, 2008·Current Protocols in Molecular Biology·S Tabor
Nov 7, 2009·Methods in Enzymology·Alois Jungbauer, Rainer Hahn
Sep 4, 2010·Current Protocols in Protein Science·Dana M Francis, Rebecca Page
Jun 14, 2012·Protein Science : a Publication of the Protein Society·Yunhua ShiBryan F Shaw
Sep 6, 2012·Advances in Experimental Medicine and Biology·Jacqueline M Matthews, Margaret Sunde
Dec 12, 2012·Annual Review of Physical Chemistry·Clement E Blanchet, Dmitri I Svergun
Nov 12, 2013·FEBS Letters·Mario Lebendiker, Tsafi Danieli
May 27, 2014·Frontiers in Microbiology·Germán L Rosano, Eduardo A Ceccarelli
Feb 1, 2017·Chembiochem : a European Journal of Chemical Biology·Kalli C CatcottZhaohui Sunny Zhou
Mar 28, 2017·Analytical Chemistry·Jinrui GanMichal Sharon
Jan 30, 2018·Journal of Proteome Research·Marshall BernAlbert J R Heck
Apr 28, 2018·Science·Gavin YoungPhilipp Kukura
May 4, 2018·Nucleic Acids Research·Anna PerederinaAndrey S Krasilnikov
Jul 4, 2019·Methods in Molecular Biology·Daniel Some, Vladimir Razinkov
Jul 10, 2019·Journal of Visualized Experiments : JoVE·Daniel SomeMario Lebendiker
Jan 12, 2020·Communications Biology·Gili Ben-NissanMichal Sharon
Jan 17, 2020·Nature Protocols·Shay VimerMichal Sharon
Feb 10, 2020·Journal of Molecular Biology·Tobias W Gräwert, Dmitri I Svergun
Mar 14, 2020·Angewandte Chemie·Fabian SoltermannWeston B Struwe
Apr 15, 2020·Nature Communications·Adar Sonn-SegevPhilipp Kukura
Apr 25, 2020·Analytical and Bioanalytical Chemistry·Kotaro TakanoSatoko Akashi
Aug 7, 2020·Nucleic Acids Research·Yiwen LiPhilipp Kukura

❮ Previous
Next ❯

Citations

Aug 19, 2021·Chemical Reviews·Rivkah Rogawski, Michal Sharon
Aug 28, 2021·Chemical Reviews·Jack L BennettWilliam A Donald

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.