On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies.
Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The non-covalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS.
Benkestock
Kurt
K
Biovitrum AB, Chemical Technologies, SE-112 76 Stockholm, Sweden. kurt.benkestock@biovitrum.com
Edlund
Per-Olof
PO
Roeraade
Johan
J
D000818:Animals; D001667:Binding, Competitive; D003570:Cytidine Triphosphate / Q000032:analysis; D006736:Horses; D008024:Ligands; D017551:Microdialysis / Q000295:instrumentation*; D009211:Myoglobin / Q000032:analysis; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D008024:Ligands; D009211:Myoglobin; D003570:Cytidine Triphosphate; D012259:Ribonuclease, Pancreatic