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Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva.

ABSTRACT: The influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique.The application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments.The use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

SUBMITTER: Gutierrez A

PROVIDER: S-EPMC5716369 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva.

Gutiérrez Ana A Cerón José Joaquín JJ Razzazi-Fazeli Ebrahim E Schlosser Sarah S Tecles Fernando F

BMC veterinary research 20171204 1

<h4>Background</h4>The influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Pr ...[more]

PMID: 29202764

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Project description:In this study, the influence of several factors such as breed, sex, and production stage over the normal range values of salivary biomarkers of health status was evaluated in pigs. A total of 409 pigs of 2 different breeds (conventional Large White × Duroc and Iberian pigs) were included in the study. Animals were divided into different groups according to their sex (male or female) and the stage of the production cycle they were in (post-weaning, nursery, fattening, and finishing). The levels of an inflammatory marker, adenosine deaminase (ADA), and two acute phase proteins, C-reactive protein (CRP) and haptoglobin (Hp) were measured in saliva samples. Moreover, the total antioxidant capacity level (TAC) was quantified for the first time in porcine saliva; therefore, an analytical validation and stability analysis during storage at -80°C were also performed. Differences according to breed were observed for all the markers studied; thus, the influence of age and sex on the normal range values were studied separately for conventional and Iberian pigs. In Large White × Duroc pigs the overall median values of ADA, CRP, Hp and TAC were 282 U/L, 10.49 ng/mL, 0.88 μg/mL, and 21.73 μM Trolox equivalents, respectively. However, higher values of inflammatory marker and acute phase proteins were observed in males at the initial stages of the production cycle, while females presented higher values when they had reached sexual maturity. In Iberian pigs the overall median values observed were 585 U/L, 4.81 ng/mL, 0.63 μg/mL, and 21.21 μM Trolox equivalents for ADA, CRP, Hp, and TAC respectively with slight differences in the influence of the studied factors. Sex differences were not observed in the levels of acute phase proteins in Iberian pigs, probably due to the castration of males during the first days of life; however, ADA levels were found to be higher in male pigs at the end of the production cycle. It could be concluded that breed, sex, and production stage influence the range values of salivary markers of health status in pigs and should be taken into account to further establish reference intervals.

Project description:Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.

Dataset Information

Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva.

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Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva.

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