Exploring Minor Proteins and Peptides in Human Milk: a Proteomic Analysis Across Lactation Stages

Condition(s) studied

Study summary

Human milk (HM) is the optimal food source for the nutrition, growth, and development of newborns. The protein fraction of HM plays a crucial role in the healthy development of infants. HM contains a wide variety of minor whey proteins and peptides with important bioactive functions, many of which are still unknown. Proteomics allows for the study of biological samples with inherently complex protein mixtures. Proteins are essential for the development of living organisms, both in quantitative and qualitative terms. The combination of proteomic techniques currently enables the study of protein variability and minor peptides in HM across different lactation stages and allows for differential quantification according to gestational age and birth weight. However, studies on the human milk serum proteome during these stages are limited. The aim is to explore the minor whey proteins and peptides in human milk through a longitudinal analysis of five groups of breastfeeding mothers (with 30 extremely low birth weight newborns, 30 very low birth weight newborns, 30 low birth weight newborns, 30 adequate birth weight newborns, and 30 high birth weight newborns). Gestational age will also be considered to ensure homogeneous group distribution according to this condition. HM samples will be collected from each mother during three lactation periods after birth: within the first 48 hours (colostrum), at 5-14 days (transitional milk), and at 100-120 days (mature milk) for the five birth weight groups. In these neonatal/infant groups, minor proteins from whey fraction and peptides will be separated, quantified, and identified using label-free proteomic techniques. This study aims to expand our understanding of the minor proteins and peptides in human milk and their bioactive roles in neonatal health. By examining these components across different birth weight groups and lactation stages, the research will offer insights into how protein and peptide profiles vary by gestational age and birth weight, potentially influencing neonatal development. The findings from this proteomic analysis could not only demonstrate the complexity of human milk composition but also contribute to targeted nutritional support for preterm or low-birth-weight infants, customizing protein supplementation in HM banks and therefore enhancing their growth and developmental outcomes.

Eligibility

Primary outcome measure(s)

• Minor whey proteins: Lysozyme — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Concentration of lysozyme in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
• Minor whey proteins: Lactoferrin — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Concentration of lactoferrin in mg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
• Minor whey proteins: Bile salt-dependent lipase (BSDL) — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Concentration of bile salt-dependent lipase (BSDL) in µg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
• Minor whey proteins: Lactoperoxidase — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Concentration of lactoperoxidase in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
• Minor whey proteins: Alpha-1-antitrypsin — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Concentration of alpha-1-antitrypsin in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
• Proteome — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Protein signatures will be assessed through a proteomic analysis using nano-liquid chromatography (nESI-MS/MS). The analysis will be followed by bioinformatic analysis with dedicated software to analyze and achieve the relative quantification of differential proteins. A specific database representing the human proteome, updated in the UNIPROT repository, will be used. The results will be compared against the protein databases available in international platforms such as SWISS-Prot, NCBI, EBI, and TrEMBL. The results are typically expressed in units or formats that reflect the relative abundance or quantitative values of proteins or peptides, such as spectral counts, percentage and µg/ml.
• Free peptides, primarily derived from β-casein — Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)
  Free peptides will be expressed in counts per peptide. Human milk samples will undergo trypsin digestion prior to analysis by liquid chromatography-mass spectrometry (LC-MS).

Trial sites (2)

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