ABSTRACT
Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS-acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (K(D)) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.
Subject(s)
Amyloid beta-Peptides/analysis , alpha-Synuclein/analysis , Amino Acid Sequence , Amino Acid Substitution , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/radiation effects , Animals , Antibodies, Monoclonal/metabolism , Antibody Affinity , Biosensing Techniques , Brain/metabolism , Cyclotrons , Epitopes , Feasibility Studies , Fourier Analysis , Humans , Mass Spectrometry , Mice, Transgenic , Microfluidic Analytical Techniques , Molecular Weight , Mutant Proteins/analysis , Mutant Proteins/chemistry , Mutant Proteins/radiation effects , Neurons/chemistry , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/radiation effects , Sound , alpha-Synuclein/chemistry , alpha-Synuclein/genetics , alpha-Synuclein/radiation effectsABSTRACT
Gas-phase protein separation by ion mobility: With its ability to separate the Parkinson's disease protein α-synuclein and its autoproteolytic products-despite the small concentrations of the latter-ion-mobility MS has enabled the characterization of intermediate fragments in in vitro oligomerization-aggregation. In particular, a possible key fragment, the highly aggregating C-terminal fragment, αSyn(72-140), has been revealed.
Subject(s)
Biopolymers/metabolism , Mass Spectrometry/methods , Parkinson Disease/metabolism , alpha-Synuclein/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Proteolysis , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
The antimycotic drug clotrimazole inhibits the function of the gastric H,K-ATPase in a manner similar to that observed for the Na,K-ATPase. Because of the high hydrophobicity of the compound, the interaction between clotrimazole and the ion pump occurs at the membrane domain in the apolar core of the membrane. The enzymatic activity was inhibited with a half-saturating concentration of 5.2 microM. Various partial reactions of the pump cycle were analyzed with the electrochromic styryl dye RH421 that has been widely used to study the transport mechanism of P-type ATPases. We discovered that the interaction of clotrimazole with the H,K-ATPase introduces a single "dead-end" branch added to the Post-Albers scheme in the E(1) state of the pump. In this inhibiting state, the ion binding sites have a significantly enhanced affinity for protons and bind up to two protons even at pH 8.5. Inhibition of the pump can be reversed by a decreased pH or increased K(+) concentrations. The mechanistic proposal that allows an explanation of all experiments presented is similar to that published for the Na,K-ATPase.
