MASS SPECTROMETRY AS A BIOANALYTICAL TOOL FOR PROTEOMIC AND IONOMIC STUDIES OF HUMAN MILK: ENRICHMENT OF LOW-ABUNDANCE PROTEINS AND CHARACTERIZATION OF PRETERM LACTATION

Abstract

Human milk is biologically extraordinary, carrying hundreds of immune-active proteins, enzymes, and minerals that collectively protect and shape the developing infant. For a researcher, however, this complexity presents a significant analytical challenge. A handful of highly abundant proteins casein, α-lactalbumin, lactoferrin, and secretory IgA dominate the fluid at concentrations spanning six to eight orders of magnitude above the rarer proteins that often carry the most important biological signals. This dissertation applies high-resolution mass spectrometry not simply as an analytical instrument, but as a means to look past these dominant proteins and uncover the hidden molecular landscape of human milk. The first study addresses a methodological necessity: finding a reliable, cost-effective way to see what is hidden. We conducted the first systematic head-to-head evaluation of five protein depletion strategies: centrifugation, acetone precipitation, methanol-chloroform precipitation, a commercial depletion kit (Minute ML-044), and perchloric acid (PerCA) precipitation, against five performance dimensions: total protein identification, high-abundance protein depletion efficiency, low-molecular-weight protein enrichment, unique protein identification, and cost per sample. PerCA emerged as the most efficient and cost-effective strategy at approximately one cent per sample, over 300 times less expensive than the commercial kit uniquely enriching low-molecular-weight proteins below 26 kDa (37.5% of unique identifications), identifying 93 proteins not detected by any other method, and uncovering 115 proteins not previously reported in any of four publicly available human milk proteome databases. These findings establish a validated, accessible framework that allows researchers to probe the milk proteome at a depth not previously achievable with a single depletion approach, and are published in ACS Omega 2025. Building directly on these methodological findings, the second study applies this validated framework to a clinical population that needs it most: mothers of very preterm infants (gestational age 22 to 31 weeks). By analyzing paired milk samples from eight mothers at Week 1 (postpartum days 7 to 10) and Week 4 (postpartum days 21 to 28) using two complementary preparation strategies, undepleted and PerCA-depleted, alongside elemental profiling of ten clinically relevant minerals by ICP-MS (PerkinElmer NexION 2000), we produced the first integrated proteomic and ionomic portrait of preterm milk maturation across early lactation. Across 1,043 total proteins identified, stage-specific proteomic shifts were observed in proteins involved in infection defense (ELANE and BPI, elevated at Week 4) and gut barrier formation (MUC5B, elevated at Week 1), alongside meaningful changes in clinically relevant minerals including iron, zinc, calcium, and phosphorus, findings with direct implications for NICU supplementation protocols and donor milk fortification strategies. Together, these studies advance human milk science in two important and connected ways. Methodologically, they provide a practical, affordable, and validated tool for comprehensive milk proteomics accessible to researchers in any laboratory setting. Biologically, they offer the first molecular-level insight into how the protein and mineral composition of preterm milk dynamically evolves during the most critical weeks of neonatal life, a window during which nutritional decisions directly shape long-term outcomes. Ultimately, this work is a step toward understanding human milk not as a static nutritional fluid but as a dynamic biological system, and toward improving clinical nutrition and care for the most vulnerable infants in the NICU.