Proteomics of Human Milk: Definition of a Discovery Workflow for Clinical Research Studies

Milk is a complex biological fluid
composed mainly of water, carbohydrates,
lipids, proteins, and diverse bioactive factors. Human milk represents
a unique tailored source of nutrients that adapts during lactation
to the specific needs of the developing infant. Proteins in milk have
been studied for decades, and proteomics, peptidomics, and glycoproteomics
are the main approaches previously deployed to decipher the proteome
of human milk. In the present work, we aimed at implementing a highly
automated pipeline for the proteomic analysis of human milk with liquid
chromatography mass spectrometry (MS). Commercial human milk samples
were used to evaluate and optimize workflows. Centrifugation for defatting
milk samples was assessed before and after reduction, alkylation,
and enzymatic digestion of proteins, without and with presence of
surfactants. Skimmed milk samples were analyzed using isobaric labeling-based
quantitative MS on an Orbitrap Tribrid mass spectrometer. Sample fractionation
using isoelectric focusing was also evaluated to more deeply profile
the human milk proteome. Finally, the most appropriate workflow was
transferred to a liquid handling workstation for automated sample
preparation. In conclusion, we have defined and describe herein an
efficient highly automated proteomic workflow for human milk sample
analysis. It is compatible with clinical research, possibly allowing
the analysis of sufficiently large cohorts of samples.