Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites.
Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein-ligand and protein-protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein-ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa alpha-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein-spermine noncovalent interaction. Spermine binding is localized to residues 106-138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets.
Xie
Yongming
Y
Department of Chemistry and Biochemistry and Department of Biological Chemistry, University of California--Los Angeles, Los Angeles, California 90095, USA.
Zhang
Jennifer
J
Yin
Sheng
S
Loo
Joseph A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D011506:Proteins