Human milk is the most complete and ideal form of nutrition for the developing infant. The composition of human milk consistently changes throughout lactation to meet the changing functional needs of the infant. The human milk proteome is an essential milk component consisting of proteins, including enzymes/proteases, glycoproteins, and endogenous peptides. These compounds may contribute to the healthy development in a synergistic way by affecting growth, maturation of the immune system, from innate to adaptive immunity, and the gut. A comprehensive overview of the human milk proteome, covering all of its components, is lacking, even though numerous analyses of human milk proteins have been reported. Such data could substantially aid in our understanding of the functionality of each constituent of the proteome. This review will highlight each of the aforementioned components of human milk and emphasize the functionality of the proteome throughout lactation, including nutrient delivery and enhanced bioavailability of nutrients for growth, cognitive development, immune defense, and gut maturation.

Background: Numerous studies have investigated the effects of the supplementation of fructooligosaccharides (FOS) on the number of bacteria in the gut that are good for health, but the results have been inconsistent. Additionally, due to its high fermentability, supplementation of FOS may be associated with adverse gastrointestinal symptoms such as bloating and flatulence. Therefore, we assessed the effects of FOS interventions on the composition of gut microbiota and gastrointestinal symptoms in a systematic review and meta-analysis. Design: All randomized controlled trials published before 10 July 2022 that investigated the effects of FOS supplementation on the human gut microbiota composition and gastrointestinal symptoms and met the selection criteria were included in this study. Using fixed or random-effects models, the means and standard deviations of the differences between the two groups before and after the intervention were combined into weighted mean differences using 95% confidence intervals (CIs). Results: Eight studies containing 213 FOS supplements and 175 controls remained in this meta-analysis. Bifidobacterium spp. counts significantly increased during FOS ingestion (0.579, 95% CI: 0.444–0.714) in comparison with that of the control group. Subgroup analysis showed greater variation in Bifidobacterium spp. in adults (0.861, 95% CI: 0.614–1.108) than in infants (0.458, 95% CI: 0.297–0.619). The increase in Bifidobacterium spp. counts were greater in the group with an intervention duration greater than 4 weeks (0.841, 95% CI: 0.436–1.247) than an intervention time less than or equal to four weeks (0.532, 95% CI: 0.370–0.694), and in the group with intervention doses > 5 g (1.116, 95% CI: 0.685–1.546) the counts were higher than those with doses ≤ 5 g (0.521, 95% CI: 0.379–0.663). No differences in effect were found between FOS intervention and comparators in regard to the abundance of other prespecified bacteria or adverse gastrointestinal symptoms. Conclusions: This is the first meta-analysis to explore the effect of FOS on gut microbiota and to evaluate the adverse effects of FOS intake on the gastrointestinal tract. FOS supplementation could increase the number of colonic Bifidobacterium spp. while higher dose (7.5–15 g/d) and longer duration (>4 weeks) showed more distinct effects and was well tolerated.

Glycopeptide-centric mass spectrometry has become a popular approach for studying protein glycosylation. However current approaches still utilize fragmentation schemes and ranges originally optimized and intended for the analysis of typically much smaller unmodified tryptic peptides. Here we show that by merely increasing the MS/MS m/z range from 2000 to 4000 during EThcD fragmentation a wealth of highly informative c and z ion fragment ions are additionally detected facilitating improved identification of glycopeptides. We demonstrate the benefit of this extended mass range on various classes of glyopeptides containing phosphorylated, fucosylated, and/or sialylated N-glycans. We conclude that the current software solutions for glycopeptide identification also require further improvements to realize the full potential of extended mass range glycoproteomics. To stimulate further developments, we provide datasets containing all classes of glycopeptides; high mannose, hybrid, and complex, measured with standard (2000) and extended (4000) m/z range that can be used as test cases for future development of software solutions enhancing automated glycopeptide analysis.

Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases.

Protein N-glycosylation on human milk proteins assists in protecting the infant's health and functions amongst others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother’s milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Here we describe an automated platform using HILIC-based cartridges enabling the proteome-wide monitoring of intact N-glycopeptides using just a digest of 150 μg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomic approach outlined above, future studies are warranted to reveal the potential impact of observed glycosylation microheterogeneity on the healthy development of infants.

Fetuin, also known as α-2-HS-glycoprotein (gene name: AHSG), is one of the more abundant glycoproteins secreted into the bloodstream. There are two frequently occurring alleles of human AHSG, resulting in three genotypes (AHSG*1, AHSG*2, and heterozygous AHSG1/2). The backbone amino acid sequences of fetuin coded by the AHSG*1 and AHSG*2 genes differ in two amino acids including one known O-glycosylation site (aa position 256). Although fetuin levels have been extensively studied, the originating genotype is often ignored in such analysis. As fetuin has been suggested repeatedly as a potential biomarker for several disorders, the question whether the gene polymorphism affects the fetuin profile is of great interest. In this work, we describe detailed proteoform profiles of fetuin, isolated from serum of 10 healthy and 10 septic patient individuals and investigate potential glycoproteogenomics correlations, e.g. how gene polymorphisms affect glycosylation. We established an efficient method for fetuin purification from individuals9 serum using ion-exchange chromatography. Subsequently, we performed hybrid mass spectrometric approaches integrating data from native mass spectra and peptide-centric MS analysis. Our data reveal a crucial effect of the gene polymorphism on the glycosylation pattern of fetuin. Moreover, we clearly observed increased fucosylation in the samples derived from the septic patients. Our serum proteoform analysis, targeted at one protein obtained from 20 individuals, exposes the wide variability in proteoform profiles, which should be taken into consideration when using fetuin as biomarker. Importantly, focusing on a single or few proteins, the quantitative proteoform profiles can provide, as shown here, already ample data to classify individuals by genotype and disease state. Graphical Abstract Highlights Frequent genetic polymorphism affects the glycosylation pattern of fetuin. Personalized in-depth proteoform profiling of fetuin purified from 20 donors. Classification of serum donors into three different genotypes. Septic patients show increased level of fucosylation at N-glycolation site N176.