Development of a Multiplexed Microcapillary Liquid Chromatography System for High-Throughput Proteome Analysis

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Abstract
Comprehensive proteome analysis requires the identification (and quantification) of the proteins in samples consisting of thousands of proteins spanning a range of abundance of several orders of magnitude. The currency of proteome analysis by mass spectrometry is the peptides generated by protein proteolysis. The high sample complexity of such samples requires a large separation capacity, which is commonly achieved by fractionation of the mixture followed by further serial separations of each fraction. The sample throughput of proteome analysis is therefore limited by the need to sequentially process large numbers of samples. We have developed a novel fourplexed microcapillary liquid chromatography system for automated, high-throughput separation of complex peptide samples. The system supports the concurrent separation of four different samples by directing identically split solvent−gradient flows into four microcapillary C18 columns. The simple design of the system achieves multiplexed separation without the need for extra solvent pumps. Peak resolution, reproducibility, and parallel separating capacity of the system were investigated using standard peptides. The applicability of the system to high-throughput protein expression profiling was demonstrated in qualitative and quantitative analyses of protein expression in S. cerevisiae grown on two different carbon sources using the isotope-coded affinity tag (ICAT) reagent and matrix-assisted desorption/ionization quadrupole time-of-flight mass spectrometry.