Multistage Ultraviolet Photodissociation Mass Spectrometry To Characterize Single Amino Acid Variants of Human Mitochondrial BCAT2
| Autor: | Jennifer S. Brodbelt, Carol L. Nilsson, James D. Sanders, M. Rachel Mehaffey, Dustin D. Holden |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Ultraviolet Rays Electrospray ionization Protein subunit Pregnancy Proteins medicine.disease_cause Mass spectrometry Mass Spectrometry Article Analytical Chemistry Minor Histocompatibility Antigens Mitochondrial Proteins 03 medical and health sciences medicine Humans Transferase Binding site Transaminases chemistry.chemical_classification Mutation Binding Sites Photodissociation Amino acid 030104 developmental biology Amino Acid Substitution chemistry Pyridoxal Phosphate Biophysics |
| Zdroj: | Analytical Chemistry. 90:9904-9911 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.8b02099 |
| Popis: | 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 multi-modal 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 multi-stage 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 multi-stage MS approach for analyzing intact protein assemblies. |
| Databáze: | OpenAIRE |
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