Variation of proteomic profile during lactation in Girgentana goat milk: a preliminary study

Abstract

The knowledge of milk proteome has been greatly enhanced by technological advances in the proteomics field as the use of the two-dimensional differential in-gel electrophoresis, a gel-based approach which allowed the analysis of proteins from complex mixtures and the comparing of several protein samples in the same experiment. The aim of this study was to characterise the whole milk proteomic profile in Girgentana dairy goat breed by two-dimensional differential in-gel elecrophoresis. The obtained representative 2D whole milk proteomic map showed a general picture of the protein distributions over the pH 3-10 NL including about 100 spots, most of them organised like a spot train. Among differentially abundant spots in the three experimental conditions, milk fat globule EGF factor 8 protein, b-lactoglobulin, b-casein and serum albumin were successfully identified. The three-dyes system employed in two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis allowed us to obtain a global representation of the Girgentana whole milk proteome. These preliminary results could be used to generate a milk reference proteomics map for the Girgentana goat breed.

Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

“…Acquired gel images analyzed using Progenesis SameSpot software (Nonlinear Dynamics, Durham, NC, USA) showed that the resolution of spots across 2DE gels produced from method A was the best compared to the other two methods. Similar extraction methods adopted by recent studies on Girgentana goat's milk proteomics [36], which also utilized a combination of urea and thiourea in their extraction buffer, showed comparable protein spot resolutions to our finding here. The further addition of ampholytes (IPG buffer) in our extraction buffer for method A helped in improving the resolution of the protein spots on the 2DE gel and minimized streaking, compared to the findings by Di Gerlando et al (2019) [36].…”

“…Similar extraction methods adopted by recent studies on Girgentana goat's milk proteomics [36], which also utilized a combination of urea and thiourea in their extraction buffer, showed comparable protein spot resolutions to our finding here. The further addition of ampholytes (IPG buffer) in our extraction buffer for method A helped in improving the resolution of the protein spots on the 2DE gel and minimized streaking, compared to the findings by Di Gerlando et al (2019) [36]. We managed to produce comparable qualities of 2DE gels that we attained from method A, in terms of total protein spots and resolution, to the previous studies on Girgentana goat's milk [36] and Greek goat's and sheep's milk [60], despite using more economical and less labor-intensive 13 cm narrow pH range (pH 4-7) gels than the more expensive 18 cm wide pH range (pH 3-10) gels used in their studies.…”

“…We managed to produce comparable qualities of 2DE gels that we attained from method A, in terms of total protein spots and resolution, to the previous studies on Girgentana goat's milk [36] and Greek goat's and sheep's milk [60], despite using more economical and less labor-intensive 13 cm narrow pH range (pH 4-7) gels than the more expensive 18 cm wide pH range (pH 3-10) gels used in their studies. Narrow range strips (pH 4-7) used in our study also allowed us to separate proteins in the casein regions better than the previous studies on milk of Girgentana goats [36] and different breeds of Greek goats and sheep [60] that had used wider pH range strips (pH 3-10).…”

“…As method A produced gels with the best spot resolution and the highest total number of protein spots, method A was chosen as the method of choice. To confirm that, six spots from each breed gel extracted from method A were randomly selected from specific milk protein regions of proteins that were identified in previous studies as serum albumin, CN, β-LG, and S100 calcium-binding protein in goat's milk and cow's milk [26,[36][37][38][39][40] (Figure 4). We also chose two protein spots with significant differences between the two breeds, to be identified by MALDI-TOF/TOF mass spectrometry.…”

“…Spot 1475 level was significantly higher in Saanen milk than Jamnapari, while spot 552 was significantly more abundant in Jamnapari milk than Saanen milk (Figure 5B). [36,41]; (B) casein region [37,38]; (C) beta-lactoglobulin region [26,39], and (D) S100 calcium-binding protein region [40].…”