Personalized Profiling Reveals Donor- and Lactation-Specific Trends in the Human Milk Proteome and Peptidome

Abstract

Background Human milk is the most genuine form of personalized nutrition, whereby its nutritional and bioactive constituents support the changing needs of the growing infant. Personalized proteome profiling strategies may provide insights into maternal–infant relationships. Proteins and endogenous peptides in human milk play an important role as nutrients for growth and have distinct functionality such as immune defense. Comprehensive monitoring of all of the human milk proteinaceous components, including endogenous peptides, is required to fully understand the changing role of the human milk proteome throughout lactation. Objective We aimed to investigate the personalized nature of the human milk proteome and peptidome for individual mother–infant dyads. Methods Two individual healthy milk donors, aged 29 and 32 y and both of a normal BMI, were longitudinally observed over weeks 1, 2, 3, 4, 6, 8, 10, 12, and 16 postpartum. Milk collection was standardized. Comprehensive variations in the human milk proteinaceous components were assessed using quantitative LC-MS/MS methods. Results We longitudinally profiled the concentrations of >1300 milk proteins and 2000 endogenous milk peptides spanning 16 wk of lactation for 2 individual donors. We observed many gradual and alike changes in both donors related to temporal effects, for instance early lactation was marked by high concentrations of proteins and peptides involved in lactose synthesis and immune development. Uniquely, in 1 of the 2 donors, we observed a substantial anomaly in the milk composition, exclusively at week 6, likely indicating a response to inflammation and/or infection. Conclusions Here, we provide a resource for characterizing the lactational changes in the human milk proteome, encompassing thousands of proteins and endogenous peptides. Further, we demonstrate the feasibility and benefit of personalized profiling to monitor the influence of milk on the development of the newborn, as well as the health status of each individual mother–infant pair.

Optimization of a human milk–directed quantitative sIgA ELISA method substantiated by mass spectrometry

“…Details of subjects and the human milk samples used in this study as well as label-free quantification (LFQ) of shotgun proteomics methodologies have been extensively described previously [14,15]. Briefly, human milk samples were collected from two individual donors across weeks 1, 2, 3, 4, 6, 8, 10, 12, and 16 of lactation under standardized conditions [16].…”

“…Whole-milk proteins were extracted, reduced, alkylated, and then digested overnight, as previously reported [15]. Tryptic peptides were separated and analyzed using liquid chromatography (LC)-MS/MS; an Agilent 1290 Infinity HPLC system (Agilent Technologies, Waldbronn, Germany) was coupled to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).…”

“…Protein concentration from shotgun, label-free quantification (LFQ) MS was derived from quantified amino acid analysis. This has previously been reported [15]. Briefly, protein pellets were precipitated from whole milk samples with 20% TCA (1:1 v/v); this extraction was then used for amino acid (AA) hydrolysis to determine total AA content to derive individual protein quantification.…”

“…Briefly, the total AA concentration was used to estimate the abundance of each protein by calculating the proportion of the mean intensity of unique peptides for each protein to the total unique peptide intensity. Full descriptions and the equations used are described in detail [15].…”

“…Crude peptides had an assumed concentration of 14 nmol/well and were diluted to 1e5 fmol/μL for analysis. MS/MS analysis and database searching have been previously described [15]. Briefly, tryptic digested peptides with spiked heavy labeled peptides and Pierce™ Peptide Retention Time Calibration Mixture (PRTC) were analyzed by LC-MS/ MS using an UltiMate 3000 HPLC (Thermo Fisher Scientific, Bremen, Germany) coupled online to a Q Exactive High Field X quadrupole-Orbitrap mass spect r o m e t e r ( T h e r m o F i s h e r S c i e n t i f i c , B r e m e n , Germany).…”