Reference 1D and 2D electrophoresis maps for potential disease related proteins in milk whey from lactating buffaloes and blood serum from buffalo calves (Water buffalo, Bubalus bubalis)

Abstract

The aim of this study was to identify potential disease related proteins in milk whey of lactating buffaloes and blood serum of buffalo calves, in order to define a reference electrophoresis map for 1-DE and 2-DE. Additionally, changes in some protein patterns from buffalo calves during salmonellosis and lactating buffaloes during mastitis are presented. Milk samples were collected and distributed into groups: Milk samples from healthy buffaloes (SCC < 100.000 cells/ml, negative microbiology and CMT) (G1, n = 5) and buffaloes with subclinical mastitis (SCC > 500.000 cells/ml, positive microbiology and CMT) (G2, n = 5). Blood samples from buffalo calves (n = 6) were collected, and three calves were experimentally infected with Salmonella Dublin and samples analyzed before (M0) and 72 h after inoculation (M1). 1-DE was accomplished by loading 10 μg of TP into SDS-PAGE, stained with Coomassie blue. 2-DE was accomplished by loading 200 μg of TP into 11 cm, pH 3-10 non-linear IPG strips, followed by SDS-PAGE, stained with Coomassie blue. Protein bands/spots were excised, subjected to tryptic in-gel digestion and analyzed by LC/ESI-MS/MS. Protein identity was assigned using NCBI databases. After bands/spots from 1-DE and 2-DE were analyzed, a protein map with 35 and 40 different identified proteins in blood serum and milk whey, respectively, was generated. Significant changes in patterns of haptoglobin were observed in buffalo calves with salmonellosis and in patterns of IgLC, β-lactoglobulin and α-lactalbumin of lactating buffaloes during mastitis. The establishment of a protein map for 1-DE and 2-DE, identifying potential disease related proteins, can help to address alterations during diseases in buffaloes.

Proteomic changes in the milk of water buffaloes (Bubalus bubalis) with subclinical mastitis due to intramammary infection by Staphylococcus aureus and by non-aureus staphylococci

“…1). The major protein bands corresponding to lactoferrin, albumin, caseins, alpha-lactalbumin and beta-lactoglobulin were clearly affected 17 . Specifically, lactoferrin and albumin increased in staphylococcus-positive samples, while caseins, alpha-lactalbumin, and beta-lactoglobulin decreased.…”

“…Sparse proteomic analyses, especially when compared to cow mastitis, have been performed on this species 15,16 . A recent proteomic investigation provided useful information on the profile of buffalo milk with mastitis, but it was limited to one-dimensional and two-dimensional electrophoresis of whey followed by the identification of the main protein spots for the purpose of setting up reference maps and of identifying acute phase proteins (APP) 17 . Here, we applied a shotgun proteomics workflow combining high performance orbitrap mass spectrometry with label-free quantitation to the milk of animals with subclinical mastitis due to staphylococcal IMI and of healthy animals with the following aims: to provide a vast dataset of buffalo milk proteins, to evaluate and understand the impact of subclinical staphylococcal mastitis on the buffalo milk proteome, to assess the differential impact of SAU and NAS IMI, and to identify novel markers for improving mastitis detection.…”

Proteomic Analysis in Morquio A Cells Treated with Immobilized Enzymatic Replacement Therapy on Nanostructured Lipid Systems

“…One of the first proteomic approaches includes a one or two-dimensional polyacrylamide gel system using sodium dodecyl sulfate (1D SDS-PAGE and 2D SDS-PAGE) and provides a powerful tool to monitor protein purification and rapidly profile the global protein expression and posttranscriptional modifications [22]. Although 1D and 2D-PAGE are common proteomic approaches for high throughput screening of putative biomarkers in several disorders, particularly in animal studies [23,24], they have not been used in lysosomal diseases. On the other hand, mass spectrometry (MS) is a beneficial proteomic technology that allows proteomic pattern visualization and the identification of proteins [25,26] and their post-transcriptional modifications, such as phosphorylation, glycosylation, similar to 2D-Page approach [27].…”

Blood serum acute phase proteins and iron dynamics during acute phase response of Salmonella enterica serotype Dublin experimentally infected buffalo calves

“…Expression patterns of ceruloplasmin, β-haptoglobin, α-haptoglobin and transferrin were obtained by performing isoelectric focusing followed by SDS-PAGE, as performed by Santana et al (2018) for blood serum samples. Equal protein loading of 200 μg was used, in all samples, for 2-DE protein separation, and samples were loaded in 11 cm, pH 3-10 nonlinear IPG strips (BioRad, Hemel Hempstead, UK) according to manufacturer's instructions.…”