Enrichment of Low-Abundance and Low-Molecular-Weight Proteins in Human Milk Using Mass Spectrometry-Based Proteomics

Human milk is a uniquely complex biological fluid, rich in bioactive components such as proteins, antibodies, enzymes, and hormones that are critical for immunity, development, and disease prevention. However, studying these trace proteins requires specialized depletion methods to counteract the dominance of abundant proteins. In this study, we systematically evaluated five different protein depletion methods: centrifugation, organic solvent-based approaches, acid precipitation, and a commercial kit (CK) to assess their efficacy in enriching low-abundance proteins (LAPs) for LC-MS/MS analysis. The comparative analysis of different depletion methods selectively enriched the sequencing depth of LAPs, highlighting the necessity of method selection in milk proteomics research. Our results further revealed that perchloric acid (PerCA) precipitation was the most effective method for identifying unique low-molecular-weight proteins (LMWPs), and optimization of the abundant protein depletion strategy could greatly increase (>10%) the extraction of LMWPs from milk samples. Additionally, several new proteins were found in our investigation when compared to publicly available milk proteome data. Thus, expanding the list of human milk proteomes enriched the data set and offered valuable insights into the complex biological functions of human milk and its impact on neonatal research.