Selective detection of specific protein-ligand complexes by electrosonic spray-precursor ion scan tandem mass spectrometry.
A novel mass spectrometric method for the selective detection of specific protein-ligand complexes is presented. The new method is based on electrosonic spray ionization of samples containing protein and ligand molecules, and mass spectrometric detection using the precursor ion scanning function on a triple quadrupole instrument. Mass-selected intact protein-ligand complex ions are subjected to fragmentation by means of collision-induced dissociation in the collision cell of the instrument, while the second mass analyzer is set to the m/z of protonated ligand ions or their alkali metal adducts. The method allows for the detection of only those ions which yield ions characteristic of the ligand molecules upon fragmentation. Since the scan range of first analyzer is set well above the m/z of the ligand ion, and the CID conditions are established to permit fragmentation of only loosely bound, noncovalent complexes, the method is specific to the detection of protein-ligand complexes under described conditions. Behavior of biologically specific and nonspecific complexes was compared under various instrumental settings. Parameters were optimized to obtain maximal selectivity for specific complexes. Specific and nonspecific complexes were found to show markedly different fragmentation characteristics, which can be a basis for selective detection of complexes with biological relevance. Preparation of specific and nonspecific complexes containing identical building blocks was attempted. Complex ions with identical stoichiometry but different origin showed the expected difference in fragmentation characteristics, which gives direct evidence for the different mechanism of specific versus nonspecific complex ion formation.
Czuczy
Noemi
N
Cell Screen Applied Research Center, Semmelweis University, Budapest, Hungary.
Katona
Maria
M
Takats
Zoltan
Z
D045304:Cytochromes c / Q000737:chemistry*; D008024:Ligands; D009113:Muramidase / Q000737:chemistry*; D011485:Protein Binding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014312:Trisaccharides / Q000737:chemistry
D008024:Ligands; D014312:Trisaccharides; C008317:maltotriose; D045304:Cytochromes c; D012259:Ribonuclease, Pancreatic; D009113:Muramidase