Multistage Ultraviolet Photodissociation Mass Spectrometry To Characterize Single Amino Acid Variants of Human Mitochondrial BCAT2
- Resource Type
- Authors
- Jennifer S. Brodbelt; Carol L. Nilsson; James D. Sanders; M. Rachel Mehaffey; Dustin D. Holden
- Source
- Analytical Chemistry. 90:9904-9911
- Subject
- 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
- Language
- ISSN
- 1520-6882
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 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.