Dataset Information

Cow MFGM LC-MS/MS - Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane

ABSTRACT: Pasteurized whole cow milk (Grade A) was purchased from the supermarket. Whole cow milk was centrifuged at 3 000 g at 4°C for 30 min and the fat layer was recovered. The recovered fat was washed with PBS for 20 min at 37°C, and the resuspened mixture was centrifuged again to obtain the milk fat. The wash procedure was repeated three times. Finally, milk fat was washed with MilliQ water to recover the fat globules. Milk fat globules were solubilized with a SDS solution (100 mM Tris-HCl, pH 7.4, 100 mM DTT and 4% SDS) and then incubated in 95°C water bath for 10 min. The mixtures were centrifuged at 14 000 g for 15 min, and the protein samples were collected. S-Trap, FASP, in-gel, and traditional Chl/MeOH, as well as modified chloroform/methanol (Chl/MeOH) or acetone precipitation followed by in-solution digestion without clean-up of tryptic peptides methods were applied to characterization cow MFGM proteome.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Sheng Zhang

LAB HEAD: Sheng Zhang

PROVIDER: PXD009288 | Pride | 2018-10-19

REPOSITORIES: Pride

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Publications

Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane.

Yang Yongxin Y Anderson Elizabeth E Zhang Sheng S

Electrophoresis 20180425 18

Proteomic analysis of membrane proteins is challenged by the proteins solubility and detergent incompatibility with MS analysis. No single perfect protocol can be used to comprehensively characterize the proteome of membrane fraction. Here, we used cow milk fat globule membrane (MFGM) proteome analysis to assess six sample preparation procedures including one in-gel and five in-solution digestion approaches prior to LC-MS/MS analysis. The largest number of MFGM proteins were identified by suspen ...[more]

PMID: 29644703

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Project description:During lactation, mammary epithelial secretory cells secrete huge amounts of milk from their apical side. The major milk proteins, the caseins, are secreted by exocytosis, while milk fat globules are released by budding, enwrapped by the plasma membrane. Due to the number and large size of milk fat globules, the membrane surface needed for their secretion might exceed that of the apical plasma membrane. In order to identify the cellular compartments that may provide membrane during the budding of milk fat globules, a large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globules membranes was performed in mouse cells. The differential analysis of the protein profiles of these two organelles strongly suggest that, in addition to the apical plasma membrane, at least the endoplasmic reticulum, the secretory vesicles containing caseins, and potentially the mitochondria contribute to the formation of the milk fat globule membrane. Moreover, the specific analysis of the membrane-associated as well as the raft-associated proteins reinforces this possibility and points to a role for lipid rafts in milk product secretion. The subcellular localization of several major proteins of the cytoplasmic lipid droplets or of the milk fat globule membrane was investigated by immunofluorescence in lactating mammary epithelial cells. Furthermore, the localization of GM1 ganglioside, a known marker of lipid rafts and of free cholesterol, showed a clear association with both cytoplasmic lipid droplets and secreted milk fat globules. Additionally, the presence of some SNARE proteins that may be involved in casein exocytosis and of two Rab GTPases on both cytoplasmic lipid droplets and milk fat globules was demonstrated by immunofluorescence. Altogether, our results provide evidence for a pivotal role of the endoplasmic reticulum as membrane contributor to milk fat globule budding, and suggest that some SNARE proteins may spatio-temporally coordinate the secretion of milk products.

Dataset Information

Cow MFGM LC-MS/MS - Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane

Publications

Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane.

Similar Datasets

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