High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins

Abstract

In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.

Combined tissue and fluid proteomics with Tandem Mass Tags to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer's Disease case study

“…This approach, however, often results in the loss of potentially biologically relevant proteins that are bound to the high-abundance proteins being depleted whether this be a simple albumin depletion, or a depletion of the top 6-12 hyper-abundant proteins. [22,23] Using the TMTcalibrator ™ approach, albumin, for example, is still detected and measured in the CSF as the protein is still present but is not dominating the MS acquisition. In this study we detected albumin, as expected, in the six CSF samples and also observed the expected regulation between AD and control CSF samples.…”

Plasma proteomic signature of age in healthy humans

“…However, performing discovery proteomics is challenging because of the wide dynamic range of plasma proteins and because of the interference from large, multiply charged proteins such as albumin, apolipoprotein A1, and C‐reactive protein (Geyer, Holdt, Teupser, & Mann, 2017). Attempts to address this challenge by depletion of highly abundant proteins from plasma samples have generated conflicting results, with some suggestions that proteins in depleted samples are no longer representative of the those in the original sample (Bellei et al., 2011). An alternative approach is to use the SOMAscan assay, a technology that uses slow off‐rate modified aptamers (SOMAmer)‐based capture to quantify multiple proteins in human biological liquids, including plasma (Baird, Westwood, & Lovestone, 2015; Di Narzo et al., 2017; Menni et al., 2015).…”

Evolution of Proteomic Methods for Analysis of Complex Biological Samples – Implications for Personalized Medicine

“…For example it has been shown that the depletion of albumin from human plasma can also remove low abundance proteins such as cytokines (Granger et al, 2005). More recent studies (Bellei et al, 2010) have also concluded that removal of high-abundance proteins can result in a loss of non-targeted, less abundant proteins. Obviously unintentional and unknowing loss of low abundance proteins is a cause for concern in the search for biomarkers of disease.…”