The Effect of Breed, Gender, and Acid Stimulation in Dog Saliva Proteome
In conclusion, stimulation procedures and dog breed should be and smell of food  what could result in different salivary proteomes. gels in a mini- protean apparatus (BioRad) as described before . .. 4, article e, . O. D. Kothgassner, B. U. Stetina, R. Palme, B. Kepplinger, and H. Group Results. Sporting . 1/28/ Breeder: Helyne Medeiros, Jim Douthit & Julie Kempster. 8/25/ Breeder: Jessica Kalupa & Mary Beth Usery. Sire: CH Last Acre CH WIL-JOI MEET ME IN SAINT LOUIS. Dam: CH Creme's Mega Mini Me. Sire: CH Popsakadoo Deja bu Bono Dam: CH. Manager 12/16/ PM BU Mini Meet 2 - 12/16/ Boston University Track and Tennis Center Results Mixed Meter Dash.
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A scanning Molecular Dynamics densitometer with internal calibration and LabScan software GE, Healthcare were used to acquire gel images and to determine the percentage of volume of each protein band; GelAnalyzer software http: Molecular masses were determined in accordance with molecular mass standards Bio-Rad Precision Plus Protein Dual Color — run with protein samples.
Protein Precipitation Due to the limited amount of individual saliva samples, the unstimulated and acid stimulated saliva samples from each breed and gender were mixed in pools, constituting a total of 12 pools: This protocol as previously observed by us allows satisfactory results for preparation of dog saliva samples for 2-DE [ 24 ].
Then the precipitates were sonicated until total resuspension and incubated during 1 h at room temperature, being subsequently centrifuged for 5 min at rpm. Focused strips were equilibrated in two steps of 15 min each with equilibration buffer [50 mM Tris—HCl, pH 8. ImageMaster 2D Platinum v7 software was used to analyze these gel images.
Spot editing and the match were performed automatically and corrected manually. Spot volume was normalized to the total spot volume. Three laboratorial replicates of each pool were run. Protein Identification Bands and spots that differed among the factors tested were manually excised from gels and digested with trypsin following the protocol already described [ 25 ].
Each reflector MS spectrum was collected in a result independent acquisition mode, using shots per spectra in — range and fixed laser intensity to V. Searches included trypsin as digesting enzyme; peptide mass tolerance of 50 ppm; fragment mass tolerance of 0.
Peptides were only considered if the ion score indicated extensive homology. Proteins were considered if the protein score indicated significant statistical confidence. Statistical Analysis Multivariate analyse of protein bands, on one hand, and protein spots, on the other, were performed with MetaboAnalyst 3.
Data normalization was used when normal distribution was not observed, using transformation log10 or scaling methods, alone or combined. The method chosen was the one that allowed data to be normally distributed.
For univariate analysis, -test, one-way ANOVA, and two-way ANOVA were used for comparison of protein profiles band percentage volume or spots percentage volume between unstimulated and acid stimulated saliva and among breeds and genders. Discriminant variables selection was done using variable importance in the projection VIP with a threshold of 1. Finally, paired-samples -test was used for comparison of total protein concentration between saliva samples with and without stimulation.
Statistical significance was considered for. Effect of Acid Stimulation on Salivary Proteome 3. Total Protein Concentration Total protein concentration decreased significantly in stimulated saliva in males of both pure breeds Portuguese Podengo and Beagle. In females, no statistically significant differences were observed for saliva collected under the two conditions Table 2.
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Concerning salivary flow rate, although this was not measured, it was possible to observe a tendency for higher salivary flow rates in big, comparatively to small breeds and higher salivary flow rate after lemon juice induction, in all breeds.
Considering the total of the animals, 2 of the protein bands decreased F and J and one increased I1 with acid stimulation Table 3. Concerning bands F and J, the decreased levels were observed only in males and not in females.
By considering the dog breeds separately, changes induced by stimulation were observed only for Beagles: Information about mass spectrometry details of identified proteins is present in Table 4.
Although, in the pure breeds Portuguese Podengo and Greyhound, none of the individual bands from SDS-PAGE protein profiles showed statistical significant intensity differences, between the saliva collected with and without acid stimulation, the multivariate PLS-DA model clustered separately unstimulated saliva from acid stimulated saliva, in these two breeds Figure 2.
The protein bands J, K, and M were the major contributors for the differences in Greyhounds. Band M was identified as containing full-double-headed protease inhibitor, whereas the other two bands resulted in no confident identification. The protein bands C, E, and G, identified as containing IgGFc-binding protein and serum albumin, were the major contributors for differences in Portuguese Podengos Supplementary Figure 1. Scaling was applied to rows when needed; and axes show principal component 1 PC1 and principal component 2 PC2respectively, and the total variance explained by each of them.
Two-Dimensional Protein Profile 2-DE By analyzing 2-DE salivary protein profiles Figure 33 protein spots were observed to be present in lower volume in the saliva collected after stimulation: These spots were identified as serum albumin subunit A, cytoskeletal keratin, and one unknown protein Table 5. Spots excised for digestion and identification by MS are numbered.
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Total Protein Concentration The four different breeds did not differ among them for the total protein concentration of saliva, as shown by univariate statistical analysis. Also, no differences were observed between genders, neither for saliva collected without nor saliva collected with acid stimulation. Six protein bands showed a different volume among dog breeds Table 6: