Proteins

Human milk proteins are essential because they provide not only nutrition but also bioactive functions that shape infant immunity, gut development, and metabolic programming. Beyond building blocks for growth, these proteins and their derived peptides act as signals that protect against infection, modulate inflammation, and support healthy development.

Total and Targeted Proteins

The Milk Analytics Core (MAC) employs validated chemical assays to quantify total protein and uses electrochemiluminescence multiplex immunoassays on the Mesoscale Discovery (MSD) platform to precisely measure specific proteins in human milk and related matrices. MSD technology is well suited for complex samples, providing high sensitivity with minimal interference from milk components such as lipids. This approach enables absolute quantification of key bioactive proteins, including lactoferrin, osteopontin, adiponectin, calprotectin, immunoglobulins, chemokines, and cytokines.

Leveraging State-of-the-Art Proteomics

For in-depth molecular profiling, MAC collaborates with the UC San Diego Proteomics Core to deliver comprehensive proteomics, peptidomics, and post-translational modification analyses.

Assessing Protein Activity

Proteases, Lipases, and More

Human milk contains enzymes that facilitate the digestion and transformation of other milk components, including proteins, lipids, and carbohydrates. In collaboration with the O’Donoghue Lab, which specializes in high-precision enzyme assays, MAC provides quantitative analysis of proteases, lipases, and other enzymatic activities in human milk and related matrices.

Total Protein

The bicinchoninic acid (BCA) assay is used to quantify total protein in human milk by measuring the colorimetric reaction between protein-reduced copper ions and BCA under alkaline conditions. It provides a robust, reproducible estimate of total protein content and is well suited for human milk when samples are appropriately prepared to minimize interferences.

Targeted Proteins (Lactoferrin, IgA/G/M, Cytokines, and many more)

We use electrochemiluminescence multiplex immunoassay technology on the Mesoscale Discovery (MSD) platform to quantitate specific proteins in human milk or related samples. MSD assays are optimal for complex matrices as they yield low interference from other human milk components, e.g., lipids.

We have established and validated sample preparation protocols, parallelism, analytical dynamic ranges, spike recovery rates, coefficients of variation, sensitivity, and other analytical parameters for the following human proteins:

Human Lacto(trans)ferrin: quantitative determination of human lactoferrin, an iron-binding glycoprotein with potential roles in immune function, iron absorption, and gut development

Human Osteopontin: quantitative determination of human osteopontin, a highly phosphorylated glycoprotein with potential roles in immune system development, gut maturation, brain development, mineral absorption, and bone remodeling

Human Adiponectin: quantitative determination of human adiponectin, a protein with potential roles in regulating metabolism, growth, and energy balance

Human Calprotectin: quantitative determination of human calprotectin, a protein with potential roles in immune and gut development

Human Immunoglobulin A (IgA): quantitative determination of total IgA (monomeric and dimeric forms), the predominant immunoglobulin in human milk

Human Immunoglobulin G and M (IgG and IgM) Panel: quantitative determination of both total IgG and total IgM in one assay

Human Cytokine 29-Plex Panel: includes 29 biomarkers associated with inflammatory response and immune system regulation. The following chemokines and cytokines are included in the panel: Eotaxin, Eotaxin-3, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, TNF-α, TNF-β, VEGF-A

All assays and panels are validated for samples of human origin only because the assays employ human-specific antibodies. However, most assays are also available for samples from mice, rats, and other species. Please contact us if you would like to explore analyses in samples of non-human origin.

Proteomics, Peptidomics, Post-Translational Modifications (PTMs)

Human milk proteomics and peptidomics study the full range of proteins and naturally occurring peptides in human milk. Proteomics focuses on intact milk proteins and their structures, abundances, and functions, while peptidomics examines smaller peptides that are either naturally occuring or are generated by protein digestion.

Phosphoproteomics is the large-scale study of protein phosphorylation, mapping when and where phosphate groups are added to proteins to regulate signaling, metabolism, and cellular responses. More broadly, analysis of post-translational modifications (PTMs) examines chemical changes made to proteins after synthesis - such as phosphorylation, glycosylation, or acetylation - to reveal how protein function, localization, and activity are dynamically controlled.

Our proteomics, peptidomics, and PTM analyses are powered by the Thermo Orbitrap Astral mass spectrometer, combining field-leading acquisition speed with ultra-high resolution in a single platform. This enables exceptional depth of protein/peptide coverage and is build for data-independent acquisition (DIA). The platform delivers high quantitative accuracy with low coefficients of variation (~5%). The system is optimized for scale, supporting high-throughput workflows with a capacity of approximately 60-180 samples per day.

Proteases, Lipases, and More

Assessing the activity of proteases, lipases, and other digestive enzymes is offered in collaboration with the O'Donoghue lab that specializes in high-precision enzyme assays. While the core expertise lies in the quantification of protease activity, the lab also offers comprehensive detection services for lipases, cellulases, and amylases. By integrating colorimetric, fluorometric, and mass spectrometry-based platforms, we provide versatile analytical solutions. Furthermore, we leverage advanced fluorogenic activity-based probes to visualize active enzymes directly on protein gels, offering a high-resolution look at the bioactive enzyme landscape of human milk and related samples.