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

• Published in: Analytical chemistry (Washington) Vol. 90; no. 16; pp. 9904 - 9911
• Main Authors: Mehaffey, M. Rachel, Sanders, James D, Holden, Dustin D, Nilsson, Carol L, Brodbelt, Jennifer S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.08.2018
• Subjects: Amino Acid Substitution; Amino acids; Analytical chemistry; Binding Sites; Chain branching; Chemistry; Chemoresistance; Destabilization; Dimers; Dismantling; Humans; Interrogation; Ionization; Ligands; Mass spectrometry; Mass Spectrometry - methods; Minor Histocompatibility Antigens - chemistry; Minor Histocompatibility Antigens - genetics; Mitochondrial DNA; Mitochondrial Proteins - chemistry; Mitochondrial Proteins - genetics; Multistage; Mutation; Next-generation sequencing; Photodissociation; Pregnancy Proteins - chemistry; Pregnancy Proteins - genetics; Proteins; Pyridoxal Phosphate - chemistry; Spectroscopy; Transaminases - chemistry; Transaminases - genetics; Ultraviolet radiation; Ultraviolet Rays
• DOI: 10.1021/acs.analchem.8b02099
• ISSN: 0003-2700

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 implementing UVPD in a multistage MS approach for analyzing intact protein assemblies.