Protein/Peptide Fractionation

A key challenge in working with serum/plasma samples is the difficulty in observing low abundance proteins. It is estimated that 12 proteins (albumin, total IgG, a-1-antitrypsin, IgA, IgM, transferrin, haptoglobin, a-1-acid glycoprotein (orosomucoid), a-2-macroglobulin, HDL (apolipoproteins A-I & A-II) and fibrinogen) compose 96% of the protein mass in plasma (1). Immunoaffinity partitioning of these highly-abundant proteins (HAP) has proven to be an effective approach for enabling the detection of low abundance proteins (LAP). Hence, in order to delve deeper into the serum/plasma proteome, it is necessary to perform immunoaffinity depletion as a pre-fractionation step.

The first step employs immunoaffinity partitioning of the top 14 most abundant proteins (see Figure 1) prior to further analysis such as DIGE. A second experiment is then performed after both IgY14 depletion and depletion of the 81 moderately abundant proteins (MAPS) on a Supermix column (both from GenWay Biotech Inc.), thereby enabling LAP to be observed and identified. The approach is highly reproducible and robust (2). The approach has also been successfully used for urine and CSF.

• 250µl of serum/plasma is required for both the IgY14 and Supermix column immunoaffinity partitioning (GenWay Biotech. Inc.)
• 50µl is required for the IgY14 immunoaffinity portioning only
  1. Anderson, N.L., Anderson, N.G. (2002) .The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell Proteomics 1:845–867.
  2. Huang, L., Harvie, G., Feitelson, J., Gramatikoff, K., Herold, D., Allen, D., Amunngama, R., Hagler, R., Pisano, M., Zhang, W., and Fang, X. (2005) Immunoaffinity separation of plasma protein by IgY microbeads: Meeting the
     needs of proteomic sample preparation and analysis. Proteomics, 5:3314-3328.