title: Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method.
abstract: Proteomic analysis of human plasma and serum for identifying and validating disease-specific marker proteins and peptides has one major drawback besides its unique advantage as a readily available sample source for diagnostic assays. This disadvantage is represented by the predominance of several high- and middle-abundant proteins, which clearly hamper identification and quantification approaches of potential and validated protein and peptide biomarkers, which are often of very low abundance. During the last decades, a significant number of depletion and enrichment techniques evolved to address these two issues. We present here a cost-effective and easy-to-use strategy for protein depletion comprising a thermal precipitation protocol followed by a two-step liquid/liquid precipitation as well as using an immunoaffinity chromatography method for the specific enrichment and isolation of the low-abundance polypeptide N-terminal pro-B-type natriuretic peptide and its precursor proBNP clinically used as biomarkers for the detection of severe human heart failure and related diseases. The applicability of this approach is shown by SDS -CGE, SDS-PAGE, electrochemiluminescence immunoassay and nano-LC ESI-MS/MS. Our thermal precipitation protocol followed by a two-step liquid/liquid precipitation could also serve as a potential depletion technique for the characterization of other low-abundance peptides and proteins.
authors: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Medical University of Innsbruck, Austria.
authors: Bernhard
authors: B
authors: Halfinger
authors: Bettina
authors: B
authors: Sarg
authors: Arno
authors: A
authors: Amann
authors: Angelika
authors: A
authors: Hammerer-Lercher
authors: Herbert H
authors: HH
authors: Lindner