Exploring Minor Proteins and Peptides in Human Milk: a Proteomic Analysis Across Lactation Stages (PROTEOM-MILK)

Quantitative Proteomic Analysis of Minor Whey Proteins and Peptides of Human Milk Across Five Neonatal Groups Classified by Birth Weight During the Three Stages of Lactation

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.

Study Overview

• Status: Recruiting
• Conditions: Preterm; Very Low Birth Weight Preterms; Low Birthweight Infant

• Study Type: Observational
• Enrollment (Estimated): 150

Contacts and Locations

• Study Contact: Name: José Luis Gómez-Chaparro Moreno, MD, Ph.D; Phone Number: +34957736467; Email: drgomezchaparro@gmail.com
• Study Contact Backup: Name: Ángel Gil, Professor; Email: agil@ugr.es

Study Locations

• Spain
  • Córdoba, Spain, 14004
    • Recruiting
    • Hospital Universitario Reina Sofia
    • Contact: Mercedes Gil Campos, MD, Ph.D; Phone Number: +34957736467; Email: mercedes_gil_campos@yahoo.es
  • Córdoba, Spain, 14004
    • Recruiting
    • Maimonides Biomedical Research Institute of Cordoba (IMIBIC)
    • Contact: Mercedes Gil Campos, MD, Ph.D; Phone Number: +34957213745; Email: mercedes_gil_campos@yahoo.es

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

Five groups of breastfeeding mothers: Group 1 (n=30) - with neonates of extremely low birth weight; Group 2 (n=30) - with neonates of very low birth weight; Group 3 (n=30) - with neonates of low birth weight; Group 4 (n=30) - with neonates of adequate birth weight; Group 5 (n=30) - with neonates of high birth weight. Additionally, gestational age will be considered to achieve a homogeneous distribution of the groups according to this condition.

Description

Inclusion Criteria:

• Healthy mothers
• With monitored pregnancies within the care area of the Reina Sofía University Hospital in Córdoba
• With newborns expected to be exclusively breastfed until 4 months

Exclusion Criteria:

• Mothers whose newborns have any of the following conditions: congenital malformation, chromosomal abnormality, hypoxia-ischemia, gastroschisis, polycythemia, hypoglycemia, sepsis, blood incompatibility
• Pathological pregnancy, pregnancy by in vitro fertilization, or multiple pregnancies
• With no plan to exclusively breastfeed until 4 months
• Under medical treatment
• Have a drug addiction
• Refuse informed consent
• Have had previous breast surgery
• Live outside the metropolitan area

Study Plan

How is the study designed?

Number of groups / cohorts

5

Cohorts and Interventions

Group / Cohort
30 extremely low birth weight; extremely low birth weight (< 1000 g)
30 very low birth weight; very low birth weight (1000-1499g)
30 low birth weight; low birth weight (1500-2499g)
30 adequate birth weight; adequate birth weight (2500-3999g)
30 high birth weight; high birth weight (4000g or >)

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Minor whey proteins: Lysozyme	Concentration of lysozyme in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).	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)
Minor whey proteins: Lactoferrin	Concentration of lactoferrin in mg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).	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)
Minor whey proteins: Bile salt-dependent lipase (BSDL)	Concentration of bile salt-dependent lipase (BSDL) in µg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).	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)
Minor whey proteins: Lactoperoxidase	Concentration of lactoperoxidase in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).	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)
Minor whey proteins: Alpha-1-antitrypsin	Concentration of alpha-1-antitrypsin in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).	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)
Proteome	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.	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, primarily derived from β-casein	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).	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)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Muscular ecography	Muscle thickness will be measured using a Phillips HD ultrasound equipment, with a high-frequency linear probe (7.5 MHz - 15 MHz), in B-mode. The thickness (expressed in mm) of the right quadriceps muscle and subcutaneous tissue will be measured, taken at the midpoint between the greater trochanter and tibial plateau: one anteroposterior in longitudinal section, another anteroposterior in transverse plane, and another perpendicular to the latter in the same plane.	After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days
Transfontanellar ecography	Brain structure and anatomy will be measured using a convex probe with a frequency range of (3.5 - 7 MHz) in B-mode. Longitudinal and transverse sections will be measured, and the thickness of the frontal cortex and the maximum thickness of the corpus callosum will be measured in millimeters (mm).	After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
General information - Mothers	Mothers will complete a general questionnaire to gather information on sociodemographic factors, pregnancy, breastfeeding, medical conditions or complications, medications, and lifestyle habits	After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days (mothers will be asked if any changes have occurred)
General information - Newborns	A general questionnaire will be completed regarding newborn characteristics, including gestational age, birth weight and length, sex, feeding type, and any pre- and/or postnatal complications or infections.	After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days (mothers will be asked if any changes have occurred)
Dietary intake and eating habits	Food Consumption Frequency Questionnaire (CFCA) at the beginning of the study will be conducted, along with a 3-day food diary during the three stages of breastfeeding.	After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days

Collaborators and Investigators

• Sponsor: Maimónides Biomedical Research Institute of Córdoba
• Collaborators: Universidad de Granada; Hospital Universitario Reina Sofia de Cordoba; Universidad de Córdoba
• Investigators: Principal Investigator: José Luis Gómez-Chaparro Moreno, MD, Ph.D, Maimonides Biomedical Research Institute of Cordoba (IMIBIC)

Study record dates

Study Major Dates

• Study Start (Actual): December 2, 2024
• Primary Completion (Estimated): January 1, 2026
• Study Completion (Estimated): December 1, 2027

Study Registration Dates

• First Submitted: January 8, 2025
• First Submitted That Met QC Criteria: January 16, 2025
• First Posted (Actual): March 25, 2025

Study Record Updates

• Last Update Posted (Actual): March 25, 2025
• Last Update Submitted That Met QC Criteria: January 16, 2025
• Last Verified: January 1, 2025

More Information

Terms related to this study

• Keywords: prematurity; lactation; whey protein; human milk; low birth weight; proteomics; gestational age; bioactive peptides
• Additional Relevant MeSH Terms: Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Body Weight; Obstetric Labor, Premature; Obstetric Labor Complications; Pregnancy Complications; Premature Birth; Birth Weight
• Other Study ID Numbers: PROTEOM-MILK

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: The datasets generated and analyzed in this study are not publicly available due to data regulations and ethical considerations. Participants consented to the use of their data exclusively by the original team of investigators, ensuring their privacy and adherence to consent agreements.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No