High Throughput Protein Fractionation and Concentration System for MALDI-MS

DESCRIPTION (provided by applicant): We describe a novel fractionation and concentration system for high throughput analysis of low abundance polypetides in serum samples by matrix-assisted laser desoption ionization (MALDI) mass spectrometry (MS). We propose to develop this polypeptide analysis system (PPAS) commercially for the rapid and reproducible purification, fractionation and concentration of low abundance polypeptides and low molecular weight proteins from blood serum. The PPAS system consists of a series of thin layers between sample reservoirs and analyte capture sites on hydrophobia capture slides. The polypeptide analytes pass through the thin layers sequentially where separation, fractionation, concentration occur. The fractionated and concentrated polypeptides are captured on the hydrophobic capture slides, each that has a different affinity for hydrophobic polypeptides. Following analyte capture, the slides can be washed free of interfering salts or other molecules that may suppress MALDI ionization of the analytes in a mass spectrometer. In the analysis step, a MALDI matix soluiton is applied directly to the samples on the capture slides and the slides are inserted directly in a MALDI-mass spectrometer for high throughput analysis of the relative abundance of polypeptides of molecular weights from 1000 to 30,000 Daltons. A separation layer prior to the fractionation/capture slides is used to remove high-abundance proteins from the polypeptides prior to their fractionantion and capture. Polypeptides are driven through the separation and fractionation/capture layers by native protein electrophoresis, according to the native charge on the proteins at any selected pH of the sample. This mode of separation allows additional fractionation according to polypeptide isoelectric point. Concentration of analytes is achieved by providing for electrophoretically focusing the polypeptides into a small capture area on the fractionion/capture slides. Phase 1 of this research project includes testing, optimization and validation of a improved fractionation system in the PPAS for analysis of polypeptides in human serum. Subsequently Phase II of this research project would entail the design and construction of a beta prototype leading to commercialization of the system.