Differential Human Plasma Proteomics Based on AniBal Quantification and Peptide-level Off-Gel Isoelectric Focussing

Abstract

Despite its enormous complexity, human plasma is still one of the most frequently used body fluids for identification and quantification of health and disease biomarkers. We have developed a new workflow for qualitative and quantitative analysis of human plasma proteins. The first step was to remove the seven most abundant plasma proteins (MARS). Moreover, in order to reduce the complexity of the sample and to increase protein and proteome coverage, Off-Gel fractionation was performed at peptide level. Our own stable isotope-based quantitative proteomics approach termed AniBAL was chosen for relative quantification of proteins between conditions. The method was developed with commercial human plasma and resulted in the identification of 85 proteins, of which 68 revealed quantitative information (Mascot database search combined with Peptide-/ProteinProphet validation). The combined methods consisting of MARS, AniBAL, Off-Gel and nano-LC-MS/MS on a Bruker HCT ion trap represent a new and efficient platform to quantify human plasma proteome differences between conditions. The method was also found technically compatible to a pair of human plasma pilot samples from the European FP6 project “DiOGenes”. Many of the identifiable/quantifiable proteins are relevant to obesity, diabetes and inflammation, which form the context of investigation within “DiOGenes”.