Abstract
Blood immerses most tissues in the body and is therefore likely to contain cell-derived proteins and peptides that may provide information about various biological processes. Serum proteome and peptidome profiling—using mass spectrometry (MS) and two-dimensionalgel electrophoresis (2-DE), for example—may thus show a functional correlate of biological events and disorders. Although blood is a convenient source of biomarkers, any study of the serum proteome is confronted with the problems. First, serum is assumed to consist of minimally tens of thousands of different protein species that span a concentration range of an estimated 10 orders of magnitude. Second, the serum proteome is dominated by a few highly abundant proteins, i.e. the 22 most abundant human serum proteins combined constitute 99% of total protein mass. Indeed almost one-half of total serum protein mass is represented by just one protein, albumin.
To overcome these problems, the sample pretreatment, clean-up, enrichment, and LC-MS conditions were evaluated. At first, serum peptides were enriched by immuno-affinity column and MWCO spin-filter. Tthe depletion of abundant proteins such as albumin, IgG and transferrin was executed with immuno-affinity column and the molecular weight based fractionation was done with MWCO filter. Serum proteins of molecular weight in 5-30kDa were analyzed with 2D-DIGE and low molecular weight proteins and peptides under 5kDa were analyzed with LC-MS/MS.
