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Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Janjanam J, Singh S, Jena MK, Varshney N, Kola S, Kumar S, Kaushik JK, Grover S, Dang AK, Mukesh M, Prakash BS, Mohanty AK - PLoS ONE (2014)

Bottom Line: Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity.The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation.The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Center, National Dairy Research Institute, Karnal, India.

ABSTRACT
Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

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Related in: MedlinePlus

2D-DIGE gel images.All 12 gels of both experiments (gels 1–6 represents early vs peak vs late lactation; gels 7–12 represents low yielding Sahiwal cows vs high yielding Sahiwal cows vs high yielding Karan Fries (KF) cross bred cows) represented above. The gels were scanned using all the three lasers corresponding to Cy2, Cy3 and Cy5 wave lengths. The images were taken at 200 µ resolution. The green color spots are down regulating and red color spots are up regulating proteins.
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pone-0102515-g004: 2D-DIGE gel images.All 12 gels of both experiments (gels 1–6 represents early vs peak vs late lactation; gels 7–12 represents low yielding Sahiwal cows vs high yielding Sahiwal cows vs high yielding Karan Fries (KF) cross bred cows) represented above. The gels were scanned using all the three lasers corresponding to Cy2, Cy3 and Cy5 wave lengths. The images were taken at 200 µ resolution. The green color spots are down regulating and red color spots are up regulating proteins.

Mentions: MECs after isolation were observed in a compound microscope. The isolated MECs looked quite homogeneous with most of the cells attaching to dynabeads (Figure 1). We observed amplification of MEC specific genes such as cytokeratin 8, α-lactalbumin and β-casein while no amplification took place in genes belonging to other cell types such as lymphocyte (CD19 & CD4) and leucocyte (CD45 & IL8) (Figure 2). The MECs from each group of animals were isolated and the proteins were extracted as shown in Figure 3. To assess the changes in protein profile in these samples, DIGE/MS analysis was performed using a pooled sample internal standard present in every gel (Figure 3B & 3D). In each experiment, every DIGE gel contained an equal aliquot of all the 12 samples labeled as Cy2 (internal standard). The proteins labeled with Cy3 and Cy5 were compared in each gel and normalized with Cy2 labeled samples. The experimental design and the DIGE analysis were followed as described by Friedman et al., 2007 [24]. From the 6 gels of each experiment a total of 2523 proteins from different stages of lactation (Gel 1–6, Figure 4) and 2672 proteins from high and low-yielding samples (Gel 7–12, Figure 4) were resolved and matched across all 6 gels of each experiment. These resolved protein features included many isoforms that resulted from charged post-translational modifications and/or processing. The differential in gel analysis (DIA) provided room for direct measurements of expression levels for each resolved protein within a gel. These spot maps were obtained from Cy3 and Cy5 channels normalized to Cy2 channel independently that eliminates technical variation due to co-migration of all tripartite labeled proteins. This methodology enabled normalization of the Cy3/Cy2 and Cy5/Cy2 intra gel ratios between gels by virtue of the signal from the Cy2 internal standard, which was present in each gel. This analysis was performed independently for the 2523 and 2672 resolved features of each experimental set. Biological variation analysis (BVA) was carried out to compare variation within the group where all the proteins were statistically significant. DIGE analysis of MEC at different stages of lactation flagged 93 protein forms and differential expression among high and low-milk yielding samples resulted in 72 protein forms with statistical significance. The changes in abundance or charge-altering post-translational modification that were greater than 1.5 fold difference includes 41 unique proteins with regard to different stages of lactation (Table 1) and 22 unique protein with regard to high and low-milk yielding groups (Table 2). Many of these unique proteins migrate as additional charge-related isoforms with indistinguishable molecular masses with charge-altering post-translational modifications. The expression patterns of these isoforms were overall similar to those listed in Table 1, 2 and in most cases these were omitted from the table for brevity. The information details of the protein identification and peptide details were listed in the supporting information tables S2 and S3. Among the 93 differentially expressed protein spots at different lactation stages, we could identify only 83 protein spots (Table S2). Whereas, among the 72 protein spots that were differentially regulated in high and low yielding samples, we could identify only 35 protein spots (Table S3). This is due to either we couldn't obtain significant spectral data or the PMF data didn't match with database for rest of these proteins. Proteins with expression changes within the 95th percentile were only considered for statistical significance. The protein spots were picked from both analytical gels and also preparative gel run with pooled protein samples that were matched with the master gel in BVA analysis. The protein spots of interest were assigned and exported to Ettan spot picker. The present analysis used medium range pH 4–7 gradients for the first dimension isoelectric focusing because this condition offers increased resolution and sensitivity compared with broader range gradients. [25].


Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Janjanam J, Singh S, Jena MK, Varshney N, Kola S, Kumar S, Kaushik JK, Grover S, Dang AK, Mukesh M, Prakash BS, Mohanty AK - PLoS ONE (2014)

2D-DIGE gel images.All 12 gels of both experiments (gels 1–6 represents early vs peak vs late lactation; gels 7–12 represents low yielding Sahiwal cows vs high yielding Sahiwal cows vs high yielding Karan Fries (KF) cross bred cows) represented above. The gels were scanned using all the three lasers corresponding to Cy2, Cy3 and Cy5 wave lengths. The images were taken at 200 µ resolution. The green color spots are down regulating and red color spots are up regulating proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128602&req=5

pone-0102515-g004: 2D-DIGE gel images.All 12 gels of both experiments (gels 1–6 represents early vs peak vs late lactation; gels 7–12 represents low yielding Sahiwal cows vs high yielding Sahiwal cows vs high yielding Karan Fries (KF) cross bred cows) represented above. The gels were scanned using all the three lasers corresponding to Cy2, Cy3 and Cy5 wave lengths. The images were taken at 200 µ resolution. The green color spots are down regulating and red color spots are up regulating proteins.
Mentions: MECs after isolation were observed in a compound microscope. The isolated MECs looked quite homogeneous with most of the cells attaching to dynabeads (Figure 1). We observed amplification of MEC specific genes such as cytokeratin 8, α-lactalbumin and β-casein while no amplification took place in genes belonging to other cell types such as lymphocyte (CD19 & CD4) and leucocyte (CD45 & IL8) (Figure 2). The MECs from each group of animals were isolated and the proteins were extracted as shown in Figure 3. To assess the changes in protein profile in these samples, DIGE/MS analysis was performed using a pooled sample internal standard present in every gel (Figure 3B & 3D). In each experiment, every DIGE gel contained an equal aliquot of all the 12 samples labeled as Cy2 (internal standard). The proteins labeled with Cy3 and Cy5 were compared in each gel and normalized with Cy2 labeled samples. The experimental design and the DIGE analysis were followed as described by Friedman et al., 2007 [24]. From the 6 gels of each experiment a total of 2523 proteins from different stages of lactation (Gel 1–6, Figure 4) and 2672 proteins from high and low-yielding samples (Gel 7–12, Figure 4) were resolved and matched across all 6 gels of each experiment. These resolved protein features included many isoforms that resulted from charged post-translational modifications and/or processing. The differential in gel analysis (DIA) provided room for direct measurements of expression levels for each resolved protein within a gel. These spot maps were obtained from Cy3 and Cy5 channels normalized to Cy2 channel independently that eliminates technical variation due to co-migration of all tripartite labeled proteins. This methodology enabled normalization of the Cy3/Cy2 and Cy5/Cy2 intra gel ratios between gels by virtue of the signal from the Cy2 internal standard, which was present in each gel. This analysis was performed independently for the 2523 and 2672 resolved features of each experimental set. Biological variation analysis (BVA) was carried out to compare variation within the group where all the proteins were statistically significant. DIGE analysis of MEC at different stages of lactation flagged 93 protein forms and differential expression among high and low-milk yielding samples resulted in 72 protein forms with statistical significance. The changes in abundance or charge-altering post-translational modification that were greater than 1.5 fold difference includes 41 unique proteins with regard to different stages of lactation (Table 1) and 22 unique protein with regard to high and low-milk yielding groups (Table 2). Many of these unique proteins migrate as additional charge-related isoforms with indistinguishable molecular masses with charge-altering post-translational modifications. The expression patterns of these isoforms were overall similar to those listed in Table 1, 2 and in most cases these were omitted from the table for brevity. The information details of the protein identification and peptide details were listed in the supporting information tables S2 and S3. Among the 93 differentially expressed protein spots at different lactation stages, we could identify only 83 protein spots (Table S2). Whereas, among the 72 protein spots that were differentially regulated in high and low yielding samples, we could identify only 35 protein spots (Table S3). This is due to either we couldn't obtain significant spectral data or the PMF data didn't match with database for rest of these proteins. Proteins with expression changes within the 95th percentile were only considered for statistical significance. The protein spots were picked from both analytical gels and also preparative gel run with pooled protein samples that were matched with the master gel in BVA analysis. The protein spots of interest were assigned and exported to Ettan spot picker. The present analysis used medium range pH 4–7 gradients for the first dimension isoelectric focusing because this condition offers increased resolution and sensitivity compared with broader range gradients. [25].

Bottom Line: Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity.The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation.The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Center, National Dairy Research Institute, Karnal, India.

ABSTRACT
Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Show MeSH
Related in: MedlinePlus