Multistage Ultraviolet Photodissociation Mass Spectrometry To Characterize Single Amino Acid Variants of Human Mitochondrial BCAT2

Analytical Chemistry
M Rachel MehaffeyJennifer S Brodbelt

Abstract

Unraveling disease mechanisms requires a comprehensive understanding of how the interplay between higher-order structure and protein-ligand interactions impacts the function of a given protein. Recent advances in native mass spectrometry (MS) involving multimodal or higher-energy activation methods have allowed direct interrogation of intact protein complexes in the gas phase, allowing analysis of both composition and subunit connectivity. We report a multistage approach combining collisional activation and 193 nm ultraviolet photodissociation (UVPD) to characterize single amino acid variants of the human mitochondrial enzyme branched-chain amino acid transferase 2 (BCAT2), a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Native electrospray ionization confirms that both proteins exist as homodimers. Front-end collisional activation disassembles the dimers into monomeric subunits that are further interrogated using UVPD to yield high sequence coverage of the mutated region. Additionally, holo (ligand-bound) fragment ions resulting from photodissociation reveal that the mutation causes destabilization of the interactions with a bound cofactor. This study demonstrates the unique advantages of implementi...Continue Reading

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Citations

Apr 25, 2019·Physical Chemistry Chemical Physics : PCCP·Sarah N Sipe, Jennifer S Brodbelt
Oct 8, 2019·Chemical Science·Jean-François GreischAlbert J R Heck
Jan 17, 2019·Biochemical Society Transactions·Timothy M Allison, Cherine Bechara
Mar 9, 2021·Journal of the American Society for Mass Spectrometry·Inês C Santos, Jennifer S Brodbelt
May 4, 2020·Journal of Proteomics·Elisabetta Boeri ErbaCarlo Petosa
Apr 24, 2021·International Journal of Mass Spectrometry·Dalton T SnyderVicki Wysocki
Aug 14, 2020·Journal of Proteome Research·Zhijie WuYing Ge
Dec 19, 2019·Chemical Reviews·Jennifer S BrodbeltInês Santos
Aug 21, 2021·Chemical Reviews·Sem TamaraAlbert J R Heck
Nov 2, 2019·Analytical Chemistry·Luis A MaciasJennifer S Brodbelt
Sep 23, 2020·Journal of the American Society for Mass Spectrometry·Alyssa Q StivingVicki H Wysocki
Aug 28, 2021·Chemical Reviews·Jack L BennettWilliam A Donald
Oct 30, 2021·Journal of the American Society for Mass Spectrometry·Luis A MaciasJennifer S Brodbelt

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