Dataset Information

Human milk protein components assessed by proteome analysis on a fast and robust micro-flow LC‒MS/MS system

ABSTRACT: Human milk is considered the best food for infants and young children, and an in-depth exploration of its molecular composition could provide a scientific basis for the development and production of substitutes. Therefore, we developed a micro-flow liquid chromatography tandem mass spectrometry (μLC‒MS/MS) system with data-independent acquisition (DIA) proteomics technology that can rapidly and stably characterize and quantify the human milk proteome.

ORGANISM(S): Homo Sapiens

SUBMITTER: Wantao Ying

PROVIDER: PXD074321 | iProX | Tue Feb 10 00:00:00 GMT 2026

REPOSITORIES: iProX

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Regional and longitudinal dynamics of human milk protein components assessed by proteome analysis on a fast and robust micro-flow LC-MS/MS system.

Cao Junxia J Cui Xinling X Lu Hai H Wang Hui H Ma Wen W Yue Zhan Z Zhen Kemiao K Wei Qiaosi Q Li Hongmei H Jiang Shilong S Ying Wantao W

Food chemistry 20241109 Pt 1

An in-depth exploration of molecular composition of human milk could provide a scientific basis for the development of substitutes. The present study was conducted to analyze human milk proteins from 110 individuals from five regions of China and across three stages of lactation to investigate the change patterns. We developed a micro-flow liquid chromatography tandem mass spectrometry (μLC-MS/MS) system with data-independent acquisition (DIA) proteomics technology that can rapidly and stably ch ...[more]

PMID: 39550967

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Project description:Background: Maternal pre-pregnancy BMI is a critical factor influencing the composition of breast milk. Breast milk has abundant extracellular vesicles (EVs) containing various biological molecules (cargo), including miRNAs. EVs are not degraded in the gastrointestinal system and circulation; thus, breast milk EVs (bEVs) interact with other organs in breastfed infants and modify the gene expression of recipient cells using miRNAs. In maternal obesity, miRNAs in bEVs are deregulated, which might be associated with adverse health outcomes in infants. In this study, we examined 798 miRNAs to determine which miRNAs are altered in the bEVs of obese mothers and their potential impact on breastfed infants. Methods: We recruited healthy nursing mothers who were either obese (BMI≥30) or lean (BMI<25) based on their pre-pregnancy BMI, and delivered a singleton baby in the prior six months. EVs were isolated from breast milk with ultracentrifugation. bEV characteristics were examined by flow cytometry and fluorescence imaging of EV markers. A total of 798 miRNAs were screened using a NanoString human miRNA panel to find deregulated miRNAs in bEVs of obese mothers compared to lean mothers. Results: We included 65 nursing mothers: 47 lean and 18 obese mothers based on pre-pregnancy BMI. After bEV isolation, we confirmed the expression of general EV markers. Out of 37 EV markers, CD326 was the most highly expressed marker in bEVs. From miRNA analysis using NanoString, we found that the most abundant miRNAs include miR-30b-5p, miR-494-3p, and let-7 families, and the list of top 10 miRNAs was not different between lean and obese mothers. Target genes of the top 10 miRNAs were associated with the EGFR, ErbB, and FoxO signaling pathway. Nineteen miRNAs were deregulated in bEVs of obese mothers (adjusted p < 0.05 cut-off), including miR-575, miR-548g-3p, miR-582-3p, and miR-652-5p. The target genes of these miRNAs are associated with lipid metabolism, inflammatory diseases, and nervous/cardiovascular system development. Conclusion: In this study, we demonstrated altered miRNAs in bEVs of obese mothers and identified the pathways of their potential target genes. Our findings will provide insight for future studies investigating the role of bEVs in breastfed infants.

Project description:Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, the transcriptome derived from the milk fat layer has not been compared with the mammary-derived transcriptome nor have sources of RNA been quantified in milk. In this study the effects of milk collection and processing on RNA quality and origin were assessed in humans and rhesus macaques. Total RNA in milk was quantitated in acridine orange-stained milk using an automated whole slide scanner and custom-built Globulator software. Total RNA extracted from milk fat, cells in milk, and mammary biopsies of lactating rhesus macaques were compared using RNA sequencing and analysis. Compared with human milk, milk from macaques contained similar amounts of RNA-containing cytoplasmic crescents, but more cells. Total RNA extracted from milk fractions was also evaluated for factors that affect RNA quality. Degradation of RNA extracted from human milk fat was positively correlated with geographic distance from collection site, storage time, and sample type. There were no differences in RNA degradation in macaque milk collected after 10 min or 4 hr accumulation, suggesting that degradation of RNA extracted from milk fat may not occur in the mammary gland. Using RNA-Seq, RNA extracted from macaque milk fat and cells in milk more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from mammary epithelial cells. Analysis of highly abundant putative microRNAs in macaque milk fat revealed a potentially novel non-coding RNA species that is conserved in humans. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells. However, this sample type clearly requires protocols that minimize RNA degradation.

Dataset Information

Human milk protein components assessed by proteome analysis on a fast and robust micro-flow LC‒MS/MS system

Publications

Regional and longitudinal dynamics of human milk protein components assessed by proteome analysis on a fast and robust micro-flow LC-MS/MS system.

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