Abstract
The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies.
Highlights
The plasma proteome has been extensively investigated with the aim of biomarker development [1, 2]
We previously reported the utility of combining multidimensional chromatography and 2D-DIGE for intact plasma proteomics
Mass spectrometric protein identification revealed that protein spots with a significant difference between the sample groups corresponded to high- and mediumabundance proteins such as acute-phase proteins, but no known plasma tumor markers were detected
Summary
The plasma proteome has been extensively investigated with the aim of biomarker development [1, 2]. Because proteins released by tumors, early-stage tumors, are expected to exist in very low concentrations and plasma contains various proteins with considerable heterogeneity between and within patients, the identification of novel plasma biomarkers represents a substantial challenge. Global expression studies on intact plasma proteins are of special interest in biomarker studies as the intact proteins reflect the functional features of protein structure. Those include posttranslational processing such as phosphorylation and glycosylation. Peptide subsets from complex digests have been analyzed for plasma proteomics, resulting in the identification of low-abundance proteins such as tissue leakage proteins [3] and biomarker candidates [4]. Much effort has been devoted to detect trace intact proteins in complex plasma samples
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