Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography

Abstract

Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.

Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data

“…This characteristic can be utilized to improve detectability of low abundance proteins by systematic depletion of high abundance proteins 58 . For example, immuno-depletion spin columns, immuno-depletion-LC and magnetic beads have been commercialized to remove up to the 20 most abundant plasma proteins [59][60][61][62][63][64] . This strategy has also been extended to deplete moderately abundant proteins (e.g., complement proteins, fibronectin, plasminogen) using Affibody molecules, beadbound peptide hexamers, or antibodies 6560,66,67 .…”

“…This characteristic can be utilized to improve detectability of low abundance proteins by systematic depletion of high abundance proteins . For example, immunodepletion spin columns, immunodepletion-LC, and magnetic beads have been commercialized to remove up to the 20 most abundant plasma proteins. − This strategy has also been extended to deplete moderately abundant proteins (e.g., complement proteins, fibronectin, plasminogen) using Affibody molecules, bead-bound peptide hexamers, or antibodies. ,,, In addition to depletion strategies using affinity reagents, methods utilizing nanoparticles also exist. ,, These nanoparticles are designed to distinguish proteins based on physical properties and can be tuned to both exclude (i.e., deplete albumin) or enrich (i.e., capture small proteins or specific analytes) simultaneously. While depletion methods can help access lower abundance plasma proteins, using these reagents has limitations, including increased sample handling, lower reproducibility/throughput, and carry-over concerns (i.e., when depleting multiple samples consecutively) .…”

Preparation of mouse anti‑human rotavirus VP7 monoclonal antibody and its protective effect on rotavirus infection

“…Due to differences in the hydrophobicity of different proteins, altering the salt concentration allows for protein precipitation. The RV VP7 monoclonal antibody precipitates in ammonium sulfate with a concentration range of 30-50%, which allows for the removal of contaminating proteins (29,30). This purification method stabilizes the antibody, reduces the risk of antibody activity loss, removes the majority of contaminating proteins and concentrates the sample (31).…”

Clinical Proteomics of Biofluids in Haematological Malignancies

“…One of the most popular immunoaffinity kits, Multiple Affinity Removal Column Human 14 (MARS14), removes the 14 most abundant proteins from human plasma and has also been used for HAP depletion of other biological fluids [ 23 ]. Furthermore, combining ammonium sulfate precipitation and protein A affinity chromatography was found to be an affordable and effective method of plasma HAP removal [ 24 ]. A recent comparison of chemi-proteomic affinity-based probes against the popular MARS14 and ProteoMiner techniques revealed the affinity capture enrichment as an effective alternative to current methods with several advantages associated with the use of these probes [ 25 ].…”