Abstract

Recently, we developed and implemented an intact protein separation scheme based on molecular weight known as gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) as the first dimensional protein fractionation. This molecular weight-based separation involves continuous elution SDS-PAGE in a tube format, in which proteins are constantly eluted from the gel column and collected in the solution phase, fractionating proteins from 5kDa to 200kDa in 1.5h. GELFrEE provided broad mass range separation with high resolution, reproducibility, and recovery, facilitating high-throughput proteomic analysis. GELFrEE fractions generated from HeLa S3 cells were successfully analyzed by online nanocapillary reversed-phase liquid chromatography (RPLC) with 12 Tesla Fourier transform mass spectrometry (FTMS) based on top-down approach. Based on our previous results for proteomic analysis, the use of GELFrEE followed by online RPLC and Orbitrap mass spectrometer with ultra-high resolution is expected to establish a high-throughput method of analyzing the peptide hormones and protein drugs covering wide molecular weight ranges. Currently, we are analyzing protein standard mixtures from 5 to 30 kDa based on top-down approach by use of online RPLC followed by Orbitrap mass spectrometer and optimizing the instrumental parameters in order to differentiate the protein drugs from endogenous forms. We will apply GELFrEE scheme to fractionate the substances present in urine samples and analyze the spiked protein drugs by use of Orbitrap mass spectrometer as the next step, thus ultimately aiming to establish a robust method of peptide and protein drugs based on MS. Einleitung