Quantitative assessment of human serum high‐abundance protein depletion

Abstract

The aim of this study is to quantify the effectivity of the depletion of human high-abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2-DE and bottom-up shotgun MS combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high-abundance protein depletion. Nano-liquid chromatography linked MS allowed the efficient identification of several low-abundant proteins that were not identified on the 2-DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.

Mass spectrometry of peptides and proteins from human blood

“…There may well be low affinity interactions by many proteins with the lipids on the surface of albumin. Recent experiments that claim to exploit albumin binding lacked control comparisons to demonstrate the proportion of binding that results from low-affinity interactions with albumin and the proportion of non-specific interactions with the chromatography resin (Lopez et al, 2005;Lowenthal et al, 2005;Stempfer et al, 2008;Lai et al, 2009). Controlled experiments to date have not conclusively shown high affinity interacting proteins associated with albumin (Marshall et al, 2003Zhang et al, 2004;Tucholska et al, 2007Tucholska et al, , 2009Williams et al, 2007) analogous to those clearly demonstrated with apolipoproteins .…”

“…Considering the capacity of a typical nano-LC column coupled with LC-MS/MS is on the order of 1 mg or less, the dilute product is still sufficient for direct analysis even after separation of digested peptides. There is evidence of non-specific binding of proteins to these depletion columns (Stempfer et al, 2008) with mixed results regarding the reproducibility of depletion column (Dekker et al, 2007;Seam et al, 2007). There is still a concentration range of 7-8 orders of magnitude after elimination of high abundance proteins (Linke, Doraiswamy, & Harrison, 2007).…”

“…Protein A from S. Aureus (Forsgren & Sjoquist, 1966) and the streptococcal bacterial protein G may interact with the Fc region of IgG (Uhlen et al, 1984), and perhaps albumin, itself (Akerstrom, Nielsen, & Bjorck, 1987), to prevent bacterial recognition by the immune system (Kronvall et al, 1979). Antibodies against albumin, or Protein G or A supports, have been used to deplete blood fluid of these abundant proteins (Gupalova et al, 2002;Shen et al, 2005;Jmeian & El Rassi, 2007;Stempfer et al, 2008). Immunodepletion columns that use multiple antibodies IgG, or IgY bound to chromatographic media have been reported to show lower background binding than dye binding resins (Pieper et al, 2003b;Echan et al, 2005;Huang & Fang, 2008;Zolotarjova et al, 2008).…”

“…Removing high abundance proteins in plasma or serum decreases the dynamic range of protein concentration in blood by two or three orders of magnitude, allowing the detection of lower abundance proteins by mass spectrometry (Roche et al, 2009). The loading capacity of most commercially available multiple depletion columns are from 8 to 35 ml of blood fluid (Stempfer et al, 2008). Hence approximately 0.5-2 mg of protein are the required starting material.…”

Extracellular Protein Phosphorylation, the Neglected Side of the Modification

“…Depletion of the highly abundant proteins was also found detrimental in the phosphoproteome analysis of CSF (26). Indeed, off-target removal was reported using albumin-specific and other immunodepletion kits (31)(32)(33). This may be because of the role of albumin as a cargo for various compounds or a result of nonspecific binding to the affinity matrix.…”