A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome.

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

O-linked glycosylations in human milk casein and major whey proteins during lactation.

As glycosylations are difficult to analyze, their roles and effects are poorly understood. Glycosylations in human milk (HM) differ across lactation. Glycosylations can be involved in antimicrobial activities and may serve as food for beneficial microorganisms. This study aimed to identify and analyze O-linked glycans in HM by high-throughput mass spectrometry. 184 longitudinal HM samples from 66 donors from day 3 and months 1, 2, and 3 postpartum were subjected to a post-translational modification specific enrichment-based strategy using TiO2 and ZrO2 beads for O-linked glycopeptide enrichment. ß-CN was found to be a major O-linked glycoprotein, additionally, αS1-CN, κ-CN, lactotransferrin, and albumin also contained O-linked glycans. As glycosyltransferases and glycosidases are involved in assembling the glycans including O-linked glycosylations, these were further investigated. Some glycosyltransferases and glycosidases were found to be significantly decreasing through lactation, including two O-linked glycan initiator enzymes (GLNT1 and GLNT2). Despite their decrease, the overall level of O-linked glycans remained stable in HM over lactation. Three different motifs for O-linked glycosylation were enriched in HM proteins: Gly-Xxx-Xxx-Gly-Ser/Thr, Arg-Ser/Thr and Lys-Ser/Thr. Further O-linked glycan motifs on ß-CN were observed to differ between intact proteins and endogenous peptides in HM.

Storage Stability of Plant-Based Drinks Related to Proteolysis and Generation of Free Amino Acids.

The market for plant-based drinks (PBDs) is experiencing a surge in consumer demand, especially in Western societies. PBDs are a highly processed food product, and little is known about this relatively new food product category when compared to bovine milk. In the present study, the storage stability, proteolysis and generation of free amino acids were investigated in commercially available PBDs over the course of a one-year storage period. Generally, pH, color and protein solubility were found to be stable in the PBDs during storage, except for the pea-based product, which showed less protein solubility after storage. The pea-based drinks also had higher initial levels of free N-terminals prior to storage compared with levels for the other plant-based drinks, as well as significantly increasing levels of total free, and especially bitter free, amino acids. The development of free amino acids in the oat-based drink indicated that the released amino acids could be involved in various reactions such as the Maillard reaction during the storage period.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Fingerprinting of Proteases, Protease Inhibitors and Indigenous Peptides in Human Milk.

The presence of proteases and their resulting level of activity on human milk (HM) proteins may aid in the generation of indigenous peptides as part of a pre-digestion process, of which some have potential bioactivity for the infant. The present study investigated the relative abundance of indigenous peptides and their cleavage products in relation to the abundance of observed proteases and protease inhibitors. The proteomes and peptidomes in twelve HM samples, representing six donors at lactation months 1 and 3, were profiled. In the proteome, 39 proteases and 29 protease inhibitors were identified in 2/3 of the samples. Cathepsin D was found to be present in higher abundance in the proteome compared with plasmin, while peptides originating from plasmin cleavage were more abundant than peptides from cathepsin D cleavage. As both proteases are present as a system of pro- and active- forms, their activation indexes were calculated. Plasmin was more active in lactation month 3 than month 1, which correlated with the total relative abundance of the cleavage product ascribed to plasmin. By searching the identified indigenous peptides in the milk bioactive peptide database, 283 peptides were ascribed to 10 groups of bioactivities. Antimicrobial peptides were significantly more abundant in month 1 than month 3; this group comprised 103 peptides, originating from the ß-CN C-terminal region.

Processing-Induced Markers in Proteins of Commercial Plant-Based Drinks in Relation to Compositional Aspects.

The consumption of plant-based drinks is increasing, but they represent a product category normally with lower protein content as compared with bovine milk. Furthermore, the products are highly processed and, therefore, the proteins in this product category may carry a significant processing history. In the present study, a series of 17 freshly produced, commercially available plant-based drinks were benchmarked according to protein-quality parameters. The plant-based drinks represented different plant sources, as well as some mixed products, and were investigated relative to composition, aggregate sizes, presence of non-reducible proteins complexes, and level of processing-induced markers in the proteins. Processing-induced changes in the proteins were determined by a newly developed cocktail method, determining markers related to Maillard and dehydroalanine pathways, as well as intact lysine by triple quadrupole-multiple reaction monitoring-mass spectrometry. It was found that all drinks contained non-reducible protein complexes, but specifically, oat-based drinks represented the largest span contents of processing-induced markers within the proteins, which may relate to their inherent processing histories. Furthermore, it was shown that in products containing added sugar, Maillard reaction-related processing markers were increased over the dehydroalanine pathway.

Development in Maillard Reaction and Dehydroalanine Pathway Markers during Storage of UHT Milk Representing Differences in Casein Micelle Size and Sedimentation.

Ultra-high temperature (UHT) processing of milk can result in protein changes during storage; however, the progress of dehydroalanine (DHA) mediated protein cross-linking and Maillard reactions in relation to the sediment formation have not been investigated previously. Liquid chromatography-mass spectrometry, based on multiple reaction monitoring (MRM), was used to absolutely quantify concentrations of furosine, N-ε-(carboxyethyl)lysine (CEL), N-ε-(carboxymethyl)lysine (CML), lanthionine (LAN) and lysinoalanine (LAL) in skim milk and sediment of UHT milk produced from raw milk with either small or large casein micelles. The results showed a higher molar proportion of the advanced stage Maillard reaction products CEL and CML in the sediment, compared to early stage Maillard reaction product furosine, whereas furosine was predominant in the skim milk. Both LAL and LAN increased during storage in the skim milk phase, however only LAL was identified in the sediment. The milk pool with large native casein micelles, known to have a higher percentage of sedimentation, contained higher proportions of furosine, CEL, CML and LAL in the sediment compared to milk with smaller native casein micelles. The study demonstrates the potential contribution of processing-induced protein-protein interactions to sedimentation in UHT milk during storage.

Using singleton densities to detect recent selection in Bos taurus.

Many quantitative traits are subject to polygenic selection, where several genomic regions undergo small, simultaneous changes in allele frequency that collectively alter a phenotype. The widespread availability of genome data, along with novel statistical techniques, has made it easier to detect these changes. We apply one such method, the "Singleton Density Score" (SDS), to the Holstein breed of Bos taurus to detect recent selection (arising up to around 740 years ago). We identify several genes as candidates for targets of recent selection, including some relating to cell regulation, catabolic processes, neural-cell adhesion and immunity. We do not find strong evidence that three traits that are important to humans-milk protein content, milk fat content, and stature-have been subject to directional selection. Simulations demonstrate that because B. taurus recently experienced a population bottleneck, singletons are depleted so the power of SDS methods is reduced. These results inform on which genes underlie recent genetic change in B. taurus, while providing information on how polygenic selection can be best investigated in future studies.

Naturally Occurring Glycosidases in Milk from Native Cattle Breeds: Activity and Consequences on Free and Protein Bound-Glycans.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including ß-galactosidase, N-acetyl-ß-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Comparison of bovine milk oligosaccharides in native North European cattle breeds.

Milk oligosaccharides are of high interest due to their bioactive properties. This study is the first to characterise milk oligosaccharides from native North European cattle breeds, as represented by 80 milk samples collected from eight native breeds originated from Norway (Norwegian Doela cattle and Norwegian Telemark cattle), Sweden (Swedish Mountain cattle), Denmark (Danish Red anno 1970), Iceland (Icelandic cattle), Lithuania (native Lithuanian Black and White) and Finland (Western Finncattle and Eastern Finncattle). Using high-performance liquid-chromatography chip/quadrupole time-of-flight mass-spectrometry, 18 unique monosaccharide compositions and a multitude of isomers were identified. No N-glycolylneuraminic acid was identified among these breeds. Western Finncattle milk was most abundant in neutral, acidic and fucosylated oligosaccharides. Further, Eastern Finncattle milk was significantly higher in acidic oligosaccharides and Icelandic cattle milk significantly higher in fucosylated oligosaccharides, compared to the mean. This study highlights specific native breeds of particular interest for future exploitation of milk oligosaccharides and breeding strategies.

Short communication: Application of proteomics for characterization of caseinomacropeptide isoforms before and after desialidation.

Caseinomacropeptide (CMP) is an important polypeptide found in cheese whey that has various biological activities and functional properties. Because sialylations play an important role in the functional properties of CMP, the aim of the present work was to characterize CMP isoform heterogeneity in a commercial glycosylated CMP (gCMP) isolate using liquid chromatography- and gel-based proteomics before and after desialidation. Using 2-dimensional gel electrophoresis (2-DGE), we observed a shift in isoelectric point (pI) after enzymatic desialidation, indicating that sialylated gCMP were located at pI 3.0 to 3.1, but desialylated gCMP had repositioned to pI 3.7 to 3.9. We used liquid chromatography/mass spectroscopy (LC-ESI/MS) for further analysis of the glycan chains of gCMP. In total, we identified 19 CMP isoforms, of which 13 were glycosylated and 6 were nonglycosylated. We also detected 3 genetic variants (A, B, and E), with up to 3 glycosylations attached per gCMP. Further, we identified up to 4 isomers, reflecting different sites of glycosylation in the peptide backbone of these isoforms. The dominating gCMP isoform of genetic variant E appeared to contain 4 N-acetyl-neuraminic acid (NeuAc) residues, whereas the dominating gCMP isoforms of variants A and B appeared to contain 2 NeuAc. The identification revealed conversions of isoforms containing different combinations of genetic variants and degrees of glycosylation, sialylation, and phosphorylation to various desialylated counter-isoforms. The content of sialylated gCMP relative to the total CMP content was 37% (wt/wt), which decreased to 1.5% after sialidase treatment, indicating 96% effectivity of the desialidation. This approach can be valuable for future functionality studies specifically addressing the importance of NeuAc.

Multiplexed bovine milk oligosaccharide analysis with aminoxy tandem mass tags.

Milk oligosaccharides (OS) are a key factor that influences the infant gut microbial composition, and their importance in promoting healthy infant development and disease prevention is becoming increasingly apparent. Investigating the structures, properties, and sources of these compounds requires a host of complementary analytical techniques. Relative compound quantification by mass spectral analysis of isobarically labeled samples is a relatively new technique that has been used mainly in the proteomics field. Glycomics applications have so far focused on analysis of protein-linked glycans, while analysis of free milk OS has previously been conducted only on analytical standards. In this paper, we extend the use of isobaric glycan tags to the analysis of bovine milk OS by presenting a method for separation of labeled OS on a porous graphitized carbon liquid chromatographic column with subsequent analysis by quadrupole time-of-flight tandem mass spectrometry. Abundances for 15 OS extracted from mature bovine milk were measured, with replicate injections providing coefficients of variation below 15% for most OS. Isobaric labeling improved ionization efficiency for low-abundance, high-molecular weight fucosylated OS, which are known to exist in bovine milk but have been only sporadically reported in the literature. We compared the abundances of four fucosylated OS in milk from Holstein and Jersey cattle and found that three of the compounds were more abundant in Jersey milk, which is in general agreement with a previous study. This novel method represents an advancement in our ability to characterize milk OS and provides the advantages associated with isobaric labeling, including reduced instrumental analysis time and increased analyte ionization efficiency. This improved ability to measure differences in bioactive OS abundances in large datasets will facilitate exploration of OS from all food sources for the purpose of developing health-guiding products for infants, immune-compromised elderly, and the population at large.

Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis.

BACKGROUND: Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa from patients with acute UC using mass spectrometry-based proteomic analysis. METHODS: Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots. RESULTS: A total of 597 spots were annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase, thioredoxins and selenium binding protein). CONCLUSIONS: A distinct proteomic profile of inflamed tissue in UC patients was found. Specific proteins involved in energy metabolism and oxidative stress were identified as potential candidate markers for UC.