Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins.

An in-depth molecular characterization of the main milk proteins, caseins (CNs) and whey proteins, from Amiata donkey combining top-down proteomic analysis (LC-MS) and cDNA sequencing revealed multiple proteoforms arising from complex splicing patterns, including cryptic splice site usage and exon skipping events. Post-translational modifications, in particular phosphorylation, increased the variety and complexity of proteoforms. α-CN perfectly exemplifies such a complexity.

An improved LC-MS method to profile molecular diversity and quantify the six main bovine milk proteins, including genetic and splicing variants as well as post-translationally modified isoforms.

Here we describe a method based on Liquid Chromatography coupled with Mass Spectrometry (LC-MS) that provides an accurate determination of the six main bovine milk proteins, including allelic and splicing variants, as well as isoforms resulting from post-translational modifications, with an unprecedented level of resolution. Proteins are identified from observed molecular masses in comparison with theoretical masses of intact proteins indexed in an "in-house" database that includes nearly 3000 entries. Quantification was performed either from UV (214 nm) or mass signals.

Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides.

In a previous study on camel milk from Kazakhstan, we reported the occurrence of two unknown proteins (UP1 and UP2) with different levels of phosphorylation. Here we show that UP1 and UP2 are isoforms of camel α-CN (α-CNsv1 and α-CNsv2, respectively) arising from alternative splicing events. First described as a 178 amino-acids long protein carrying eight phosphate groups, the major camel α-CN isoform (called here α-CN) has a molecular mass of 21,906 Da.

Impact of exposure to diesel exhaust during pregnancy on mammary gland development and milk composition in the rabbit.

Exposure to fine-particulate air pollution is a major global health concern because it is associated with reduced birth weight and an increased risk of cardiovascular disease. Here we have investigated the potential for exposure to diesel exhaust during pregnancy to influence mammary gland development and milk composition. Female rabbits were therefore exposed by nose-only inhalation to either diluted diesel exhaust fumes (1 mg/m3) or clean air for 2h/day, 5 days/week, from the 3rd to the 27th days of pregnancy.

The main WAP isoform usually found in camel milk arises from the usage of an improbable intron cryptic splice site in the precursor to mRNA in which a GC-AG intron occurs.

Background: Whey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC). The organization of the eutherian WAP genes is highly conserved through evolution. It has been proposed that WAP could play an important role in regulating the proliferation of mammary epithelial cells.

Combining different proteomic approaches to resolve complexity of the milk protein fraction of dromedary, Bactrian camels and hybrids, from different regions of Kazakhstan.

Nutritional suitability of milk is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction. Hence, to go further into the evaluation of the potential suitability of non-bovine milks in human/infant nutrition it is necessary to have a detailed characterization of their protein components. Combining proven proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS) and cDNA sequencing, we provide here in depth characterization of the milk protein fraction of dromedary and Bactrian camels, and their hybrids, from different regions of Kazakhstan.

Within-breed and multi-breed GWAS on imputed whole-genome sequence variants reveal candidate mutations affecting milk protein composition in dairy cattle.

Background: Genome-wide association studies (GWAS) were performed at the sequence level to identify candidate mutations that affect the expression of six major milk proteins in Montbéliarde (MON), Normande (NOR), and Holstein (HOL) dairy cattle. Whey protein (α-lactalbumin and β-lactoglobulin) and casein (αs1, αs2, β, and κ) contents were estimated by mid-infrared (MIR) spectrometry, with medium to high accuracy (0.59 ≤ R ≤ 0.

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.

Evolution of major milk proteins in Mus musculus and Mus spretus mouse species: a genoproteomic analysis.

Background: Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains.

Translational efficiency of casein transcripts in the mammary tissue of lactating ruminants.

Caseins are essentially concentrated in the colloidal fraction of ruminant milks as highly hydrated and mineralized spherical particles, termed casein micelles. They form a group of four peptide chains (alpha(s1), beta, alpha(s2) and kappa), encoded by four structural genes (CSN1S1, CSN2, CSN1S2 and CSN3, respectively) of which the expression is regulated by lactogenic hormones. These phosphoproteins are synthesized, essentially during lactation, in the mammary epithelial cells and we show, for the first time, that their regulation is also controlled at the translational level.

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation.

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L.

Proteomic tools to characterize the protein fraction of Equidae milk.

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer.

Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis: identification of a third N-acetylglucosaminidase, AcmC.

The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins.

Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis.

A gene encoding a putative peptidoglycan hydrolase, named acmB, which is a paralogue of the major autolysin acmA gene, was identified in the Lactococcus lactis genome sequence. The acmB gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth.

Is the apocrine milk secretion process observed in the goat species rooted in the perturbation of the intracellular transport mechanism induced by defective alleles at the alpha(s1)-Cn locus?

The structural and quantitative variability of caprine alpha(s1)-casein induced by the extensive polymorphism recorded at the corresponding locus strongly influences the composition (proteins as well as lipids) and the technological behaviour of milk. Immuno-histo-chemistry studies coupled with electron microscopy analysis have shown that a dysfunction exists in the intracellular transport of caseins when alpha(s1)-casein is lacking. Casein accumulation in the endoplasmic reticulum leads to a dilation of the cisternae that could disturb the whole secretion process (including lipids).