Reversed-Phase High-Performance Liquid Chromatographic Prefractionation of Immunodepleted Human Serum Proteins to Enhance Mass Spectrometry Identification of Lower-Abundant Proteins

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Abstract
Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered “high abundance”, present at 2−70% by mass of total protein, to those considered “low abundance”, present at 10-12 M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. Therefore, in this study, we have developed a workflow toward addressing the complexity of these samples through the application of multidimensional separation techniques. The use of reversed-phase methods for the separation and fractionation of protein samples has been investigated, with the goal of developing an optimized serum separation for application to proteomic analysis. Samples of human serum were depleted of the six most abundant proteins, using an immunoaffinity LC method, then were separated under a variety of reversed-phase (RP) conditions using a macroporous silica C18 surface modified column material. To compare the qualities of the RP separations of this complex protein sample, absorbance chromatograms were compared, and fractions were collected for off-line SDS-PAGE and 2D-LC−MS/MS analysis. The column fractions were further investigated by determination of protein identities using either whole selected fractions, or gel bands excised from SDS-PAGE gels of the fractions. In either case samples underwent tryptic fragmentation and peptide analysis using MALDI−MS or LC−MS/MS. The preferred conditions for RP protein separation exhibited reproducibly high resolution and high protein recoveries (>98%, as determined by protein assay). Using the preferred conditions also permitted high column mass load, with up to 500 μg of protein well tolerated using a 4.6 mm ID × 50 mm column, or up to 1.5 mg on a 9.4 mm ID × 50 mm column. Elevated column temperature (80 °C) was observed to be a critical operational parameter, with poorer results observed at lower temperatures. The combination of sample simplification by immunoaffinity depletion combined with a robust and high recovery RP-HPLC fractionation yields samples permitting higher quality protein identifications by coupled LC−MS methods. Keywords: proteomics • pre-fractionation • reversed-phase chromatography • immunodepletion • human serum • mass spectrometry • gel electrophoresis • HPLC

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