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. 2024 Apr 4:11:1356318.
doi: 10.3389/fvets.2024.1356318. eCollection 2024.

Characterization of the plasma proteome from healthy adult dogs

Pavlos G Doulidis  1 , Benno Kuropka  2 , Carolina Frizzo Ramos  3   4 , Alexandro Rodríguez-Rojas  1 , Iwan A Burgener  1
Affiliations

Characterization of the plasma proteome from healthy adult dogs

Pavlos G Doulidis et al. Front Vet Sci. .

Abstract

Introduction: Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine.

Methods: In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose.

Results and discussion: Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

Keywords: biomarker; canine; mass-spectrometry; plasma proteomics; veterinary medicine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Illustration of the experimental setup and data processing for the canine plasma proteome analysis used in this study. The five pools were generated using a computer script to ensure the randomization of each sample. Initially, 282 proteins were detected. After removing reverse hits and contaminants, analyzed, 255 proteins remained, and after excluding proteins not identified in at least 3 out of 5 replicates of at least one experiment, a total of 181 proteins was analyzed.
Figure 2
Figure 2
Abundance rank of the identified canine plasma proteins amongst all samples. Selected proteins representing different levels of abundances are labelled: ALB, albumin; APOA1, apolipoprotein A1; HP, haptoglobin; C3, compliment 3; GC, vitamin D-binding protein; APOA4, apolipoprotein A4; APOB, apolipoprotein B; APOC2, apolipoprotein C2; FN1, fibronectin 1; IGFALS, insulin-like growth factor binding protein, Fibrinogen alpha chain; F9, coagulation factor 9; CDH5, cadherin 5.
Figure 3
Figure 3
Volcano plots (−log p-values versus log2 fold-change) of protein intensity were measured by LC-MS of canine plasma proteins from the three conditions of plasma processing in this study (A–C). Proteins with a minimum 2-fold intensity change compared to the control (log2 fold-change ≥1 or log2 fold-change ≤ −1) and a q-value ≤0.05 were considered significantly abundant. Black dots represent non-significant differentially abundant proteins, green dots show the up-represented fraction and, and red ones represent the down-represented fraction.
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Grants and funding

The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.
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