All Cats are Gray in the Dark: Enrichment/Depletion Approaches for Biomarker Discovery on Felis catus Plasma

Abstract

In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.

Identification of potential plasma protein biomarkers for feline pancreatic carcinoma by liquid chromatography tandem mass spectrometry

“…The high efficacy can be explained by the high sequence homology (80.1%) of albumin, the most abundant HAP, between humans and dogs. 14,15 Previous research has also demonstrated successful immunodepletion of targeted HAPs from canine and bovine biological fluids using the human MARS-6 device. 19,22 Immunodepletion of feline plasma samples has been attempted previously, however researchers found that none of the four human-based HAP immunodepletion devices evaluated were efficacious.…”

“…19,22 Immunodepletion of feline plasma samples has been attempted previously, however researchers found that none of the four human-based HAP immunodepletion devices evaluated were efficacious. 15 These results were surprising, given that feline and human albumin also share a high degree of sequence homology (82.4%). 15 In contrast, our study demonstrated successful removal of albumin and likely IgG from feline plasma, which may reflect an increased antibody affinity for feline albumin in the MARS-2 kit compared to the previously tested devices.…”

“…15 These results were surprising, given that feline and human albumin also share a high degree of sequence homology (82.4%). 15 In contrast, our study demonstrated successful removal of albumin and likely IgG from feline plasma, which may reflect an increased antibody affinity for feline albumin in the MARS-2 kit compared to the previously tested devices. To the authors' best knowledge, this is the first published study validating the use of a human immunoaffinity device in feline plasma prior to proteomics analysis.…”

“…By comparing the determined concentration of three pairs of plasma and flow through samples (i.e., pre-and post-immunodepletion), the average protein recovery rate was calculated. 15…”

“…Filtering of the few protein types that make up the HAPs would remove over 90% of the total protein abundance, allowing for a markedly increased chance for LAPs to be detected by proteomics techniques. 13,14,15 Multiple affinity removal system (MARS) is one of the earliest commercially available multi-component immunoaffinity depletion/ enrichment columns, and it is commonly used for immunodepletion in proteomics studies in human medicine. 16,17 Proteomics in feline pancreatic cancer has been little explored to date.…”