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The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis.

Cugno G, Parreira JR, Ferlizza E, Hernández-Castellano LE, Carneiro M, Renaut J, Castro N, Arguello A, Capote J, Campos AM, Almeida AM - PLoS ONE (2016)

Bottom Line: It is of importance to produce strategies to oppose adverse effects of SWL.The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry.Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels.

View Article: PubMed Central - PubMed

Affiliation: CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Porto, Portugal.

ABSTRACT
Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

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Separation of mitochondrial protein complexes from Majorera breed by BN-PAGE.Proteins were solubilized with the detergent DDM at a protein ratio of 1:10; 1:7.5 and 1:5 (protein: detergent, w/w). Gel stained with Coomassie Blue Colloidal. Gel bands were excised and analysed by nano-LC-MS/MS.
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pone.0151599.g001: Separation of mitochondrial protein complexes from Majorera breed by BN-PAGE.Proteins were solubilized with the detergent DDM at a protein ratio of 1:10; 1:7.5 and 1:5 (protein: detergent, w/w). Gel stained with Coomassie Blue Colloidal. Gel bands were excised and analysed by nano-LC-MS/MS.

Mentions: The solubilisation of the mitochondrial membrane protein complexes was attempted with the non-denaturant detergent DDM. Three protein:detergent (w/w) ratios (1:5, 1:7 and 1:10) were tested. The efficiency of the solubilisation was assessed by protein profiling in BN-PAGE gels (number of bands and intensity). Upon electrophoresis, similar profiles were reported for the three protein samples tested with, at least, eight protein bands (eight protein complexes) being resolved among samples (Fig 1). The molecular mass of the protein complexes in the BN-PAGE gels varied between 146 and 720 kDa or higher (Fig 1). The results indicate that DDM, within this concentration range, has no major influence in the solubilisation of membrane proteins. Moreover the results are consistent, for example, with the separation of mitochondrial membrane complexes I-V accomplished in different rat organs [44].


The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis.

Cugno G, Parreira JR, Ferlizza E, Hernández-Castellano LE, Carneiro M, Renaut J, Castro N, Arguello A, Capote J, Campos AM, Almeida AM - PLoS ONE (2016)

Separation of mitochondrial protein complexes from Majorera breed by BN-PAGE.Proteins were solubilized with the detergent DDM at a protein ratio of 1:10; 1:7.5 and 1:5 (protein: detergent, w/w). Gel stained with Coomassie Blue Colloidal. Gel bands were excised and analysed by nano-LC-MS/MS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816393&req=5

pone.0151599.g001: Separation of mitochondrial protein complexes from Majorera breed by BN-PAGE.Proteins were solubilized with the detergent DDM at a protein ratio of 1:10; 1:7.5 and 1:5 (protein: detergent, w/w). Gel stained with Coomassie Blue Colloidal. Gel bands were excised and analysed by nano-LC-MS/MS.
Mentions: The solubilisation of the mitochondrial membrane protein complexes was attempted with the non-denaturant detergent DDM. Three protein:detergent (w/w) ratios (1:5, 1:7 and 1:10) were tested. The efficiency of the solubilisation was assessed by protein profiling in BN-PAGE gels (number of bands and intensity). Upon electrophoresis, similar profiles were reported for the three protein samples tested with, at least, eight protein bands (eight protein complexes) being resolved among samples (Fig 1). The molecular mass of the protein complexes in the BN-PAGE gels varied between 146 and 720 kDa or higher (Fig 1). The results indicate that DDM, within this concentration range, has no major influence in the solubilisation of membrane proteins. Moreover the results are consistent, for example, with the separation of mitochondrial membrane complexes I-V accomplished in different rat organs [44].

Bottom Line: It is of importance to produce strategies to oppose adverse effects of SWL.The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry.Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels.

View Article: PubMed Central - PubMed

Affiliation: CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Porto, Portugal.

ABSTRACT
Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

Show MeSH
Related in: MedlinePlus