Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.
Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Graham M
GM
West
J Will
JW
Thompson
Erik J
EJ
Soderblom
Laura G
LG
Dubois
Patrick D
PD
Dearmond
M Arthur
MA
Moseley
Michael C
MC
Fitzgerald
D000595:Amino Acid Sequence; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D021984:Cyclophilin A / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D008024:Ligands; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D011506:Proteins; D025801:Ubiquitin; D008715:Methionine; D006861:Hydrogen Peroxide; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D024402:Carbonic Anhydrase II; D021984:Cyclophilin A