Phosphoproteomics of the goat milk fat globule membrane: New insights into lipid droplet secretion from the mammary epithelial cell.

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the "milk fat globule membrane" (MFGM), the triple-layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high-sensitive Q-Exactive LC-MS/MS analysis of MFGM-derived peptides to the use of an in-house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine-C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 (http://proteomecentral.proteomexchange.org/dataset/PXD001039).

Combining proteomic tools to characterize the protein fraction of llama (Lama glama) milk.

Llamas belong to the Camelidae family along with camels. While dromedary camel milk has been broadly characterized, data on llama milk proteins are scarce. The objective of this study was thus to investigate the protein composition of llama milk. Skimmed llama milk proteins were first characterized by a 2D separation technique coupling RP-HPLC in the first dimension with SDS-PAGE in the second dimension (RP-HPLC/SDS-PAGE). Llama milk proteins, namely caseins (αs1 -, αs2 -, ß-, and κ-caseins), α-lactalbumin, lactoferrin, and serum albumin, were identified using PMF. Llama milk proteins were also characterized by online LC-ESI-MS analysis. This approach allowed attributing precise molecular masses for most of the previously MS-identified llama milk proteins. Interestingly, α-lactalbumin exhibits distinct chromatographic behaviors between llama and dromedary camel milk. De novo sequencing of the llama α-lactalbumin protein by LC coupled with MS/MS (LC-MS/MS) showed the occurrence of two amino acid substitutions (R62L/I and K89L/I) that partly explained the higher hydrophobicity of llama α-lactalbumin compared with its dromedary counterpart. Taken together, these results provide for the first time a thorough description of the protein fraction of Lama glama milk.

Proteomics of the milk fat globule membrane from Camelus dromedarius.

Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D-LC-MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p-value = 1.73 × 10(-14)), translation (p-value = 1.08 × 10(-11)), lipid biosynthetic process (p-value = 6.72 × 10(-10)), hexose metabolic process (p-value = 1.89 × 10(-04)), and actin cytoskeleton organization (p-value = 2.72 × 10(-04)). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.