Multidimensional fractionation and analysis of the Human Plasma Proteome

抄録

An ultimate objective of proteomics is the understanding of complex biological systems, which can lead to new diagnostics and therapy. The first step toward this end is the profiling of protein differences between states of a proteome. One approach to proteome profiling fractionates the proteome into intact proteins with subsequent analysis for structural characterization and identification of the protein differences. This paper presents a multidimensional approach for proteome profiling that utilizes two-dimensional liquid chromatography for fractionation of the proteome followed by analysis with capillary electrophoresis (CE) and/or mass spectrometry (MS). The first-dimension separation is done by chromatofocusing over a pH range from 8.5 to 4.0, which separates proteins by pI. Fractions are collected based on pH intervals as detected by a pH monitor. The proteins are detected by UV absorbance at 280 nm. The first-dimension fractions are separated in a second dimension by high-resolution, reversed-phase chromatography with a trifluoroacetic acid - acetonitrile gradient. The proteins are detected by absorbance at 214 nm. Fractions collected from the second dimension are analyzed by CE and MS. This multidimensional fractionation and analysis approach was used to compare fasting and non-fasting human plasma. Glycoprotein content was analyzed by CE and proteins identified by MALDI-TOF MS. The removal of the six most abundant plasma proteins (albumin, IgG, transferrin, fibrinogen, IgA and IgM) with specific chicken IgY antibodies was evaluated for effects on the proteome profile. Distinct qualitative and quantitative differences in protein expression were observed between the fasting and non-fasting states of human plasma.