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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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Two-dimensional gel electrophoresis of mitochondrial proteins from Majorera breed.Identified proteins are referred in the figure by the gene identifier and the reference number from the S1 Table. Proteins were separated within the pI and molecular mass intervals 3–10 and 20–117 kDa. Proteins were stained with CCB and identified with MALDI-TOF/TOF. Detailed information of protein identification is presented in S2 Table.
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pone.0151599.g003: Two-dimensional gel electrophoresis of mitochondrial proteins from Majorera breed.Identified proteins are referred in the figure by the gene identifier and the reference number from the S1 Table. Proteins were separated within the pI and molecular mass intervals 3–10 and 20–117 kDa. Proteins were stained with CCB and identified with MALDI-TOF/TOF. Detailed information of protein identification is presented in S2 Table.

Mentions: 2DE was employed for a complementary mapping of the mitochondrial proteome (Fig 3). This conventional proteomic analysis method enables the information accomplished here to be reproduced and utilized in most of molecular and cell biology laboratories. Proteins with masses varying between 20 and 117 kDa were separated in large format gels along a pH interval of 3–10 (Fig 3). Ninety-five proteins were identified by MALDI-TOF/TOF, this higher number comparatively to BN/PAGE-SDS/PAGE could be attributed in part to the increased capability of 2DE to separate and resolve complex protein mixtures. Moreover in comparison to BN-PAGE the high resolution of 2DE has been exploited in the characterization of protein isoforms. In the present map several putative isoforms of ATP synthase subunits alpha and beta (ATP5A, ATP5B), prelamin (LMNA), protein disulfide-isomerase (PDIA), mitochondrial inner membrane protein (IMMT), NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUSF) are reported (Fig 3, square delimited proteins).


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)

Two-dimensional gel electrophoresis of mitochondrial proteins from Majorera breed.Identified proteins are referred in the figure by the gene identifier and the reference number from the S1 Table. Proteins were separated within the pI and molecular mass intervals 3–10 and 20–117 kDa. Proteins were stained with CCB and identified with MALDI-TOF/TOF. Detailed information of protein identification is presented in S2 Table.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0151599.g003: Two-dimensional gel electrophoresis of mitochondrial proteins from Majorera breed.Identified proteins are referred in the figure by the gene identifier and the reference number from the S1 Table. Proteins were separated within the pI and molecular mass intervals 3–10 and 20–117 kDa. Proteins were stained with CCB and identified with MALDI-TOF/TOF. Detailed information of protein identification is presented in S2 Table.
Mentions: 2DE was employed for a complementary mapping of the mitochondrial proteome (Fig 3). This conventional proteomic analysis method enables the information accomplished here to be reproduced and utilized in most of molecular and cell biology laboratories. Proteins with masses varying between 20 and 117 kDa were separated in large format gels along a pH interval of 3–10 (Fig 3). Ninety-five proteins were identified by MALDI-TOF/TOF, this higher number comparatively to BN/PAGE-SDS/PAGE could be attributed in part to the increased capability of 2DE to separate and resolve complex protein mixtures. Moreover in comparison to BN-PAGE the high resolution of 2DE has been exploited in the characterization of protein isoforms. In the present map several putative isoforms of ATP synthase subunits alpha and beta (ATP5A, ATP5B), prelamin (LMNA), protein disulfide-isomerase (PDIA), mitochondrial inner membrane protein (IMMT), NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUSF) are reported (Fig 3, square delimited proteins).

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