Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses.
Abstract: Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted serum to identify the protein biomarkers for silent gastric ulceration. Gastroscopy was used to identify gastric ulceration, and serum was obtained from horses with either a healthy gastric mucosa or gastric ulceration. Serum albumin was removed using the trichloroacetic acid (TCA) protein precipitation method, and this protocol was optimised by varying the concentration of TCA, type of organic solvents, ratio of serum to protein precipitation solution, and incubation times. Electrophoresis and image analysis were used to compare the amounts of albumin, immunoglobulins G (IgG), and protein degradation before and after TCA precipitation. The best protocol was chosen to remove albumin for a proteomic study (electrophoresis and mass spectrometry). The results revealed that protocol 2 (ratio of serum to solution 1:5, 10% TCA in acetone, and 90 min incubation) was the most efficient protocol to remove albumin (98%) and IgG heavy (80%) and light (98%) chains without degrading other proteins. After electrophoresis and mass spectrometry analysis, KRT1, KRT6A and KRT18 were identified as potential markers for silent gastric ulceration.
Publication Date: 2018-09-19 PubMed ID: 30250392PubMed Central: PMC6145863DOI: 10.1294/jes.29.53Google Scholar: Lookup
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- Journal Article
Summary
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This research focuses on refining a method for removing albumin, a common protein found in horse blood samples, to allow for a more effective identification of certain biomarkers associated with silent gastric ulcers in horses.
Problem Identification
- The research is based on the issue of diagnosing silent gastric ulceration in horses known to frequently occur but is difficult to detect due to the lack of explicit symptoms.
- The team of researchers noted that Proteomic studies, which involve the large-scale study of proteins, hold potential for identifying serum biomarkers for the ulcer condition. However, the presence of albumin, a very common serum protein, often overshadows less prevalent proteins that might serve as biomarkers.
Study’s Objective
- The study aimed to create an optimized protocol to remove equine serum albumin effectively without causing degradation of other proteins, thereby allowing for a clearer analysis of potential biomarkers.
- This optimized protocol was then used to identify potential protein biomarker candidates for silent gastric ulceration in horses.
Methodology
- The team used a method known as Trichloroacetic acid (TCA) protein precipitation to deplete albumin from the horse’s blood.
- The efficiency of the protocol was tested through tweaking variables such as the concentration of TCA, type of solvents used, the ratio of serum to protein precipitation solution, and the incubation times.
- Tools like Electrophoresis (a technique used to separate proteins based on their mass or charge) and image analysis were used to assess the amount of remaining albumin, antibodies, and degradation of other proteins before and after the TCA precipitation process.
Results
- The research team identified that using a 10% TCA solution in acetone, with a 1:5 serum to solution ratio and a 90-minute incubation period, was the most effective protocol. According to this protocol, albumin reduction by 98%, and Immunoglobulins G heavy and light chains by 80% and 98% respectively were observed with no apparent damage to other proteins.
- With the refined protocol, the researchers utilized electrophoresis and mass spectrometry for proteomic study, where they identified three potential markers, KRT1, KRT6A and KRT18, for silent gastric ulceration.
Conclusion
- The successfully optimized protocol has potential for broader applications, particularly in proteomic studies involving horses and possibly other animals, to improve the detection of silent or symptomatic diseases.
- Newly identified protein markers also provide important input to the understanding of silent gastric ulceration disease in horses.
Cite This Article
APA
Poltep K, Tesena P, Yingchutrakul Y, Taylor J, Wongtawan T.
(2018).
Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses.
J Equine Sci, 29(3), 53-60.
https://doi.org/10.1294/jes.29.53 Publication
Researcher Affiliations
- The Monitoring and Surveillance Centre for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
- Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
- Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
- Proteomics Research Laboratory, Genome Technology Research Unit, National Centre for Genetics Engineering and Biotechnology, Pathum Thani 12120, Thailand.
- Biomedical Teaching Organisation, Biomedical Sciences, Edinburgh Medical School, University of Edinburgh, Edinburgh, EH8 9AG, U.K.
- Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
- Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
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