The richness and diversity of the human milk proteome explored by mass spectrometry

This thesis illustrates most of the work that I have performed during my PhD studies, focusing on the human milk proteome by mass spectrometry. In Chapter 1, a general introduction is given into the human milk proteome and the MS-based approaches used throughout this thesis. The biological functionality of the human milk proteome to meet infants’ developing requirements is described, emphasizing the roles in growth, immunity and gut development. In Chapter 2, we aimed to comprehensively characterize and quantify the human milk proteome utilizing an approach combining proteomics and peptidomics in individual donors, and applied targeted proteomics, parallel reaction monitoring (PRM), and enzyme-linked immunosorbent assay (ELISA) to further confirm some of the observed changes. We demonstrated that the difference between individuals was more substantial than the differences observed over the lactation period within a single individual. We also noticed an unexpectedly strong and prompt compositional change in one donor, which indicated an inflammatory response without maternal clinical symptom but was accompanied by a short-term skin rash observed in the infant. In Chapter 3, whether non-human proteins and peptides were in human milk has been a topic of interest for decades; however, the final verdict on their presence was hindered due to cross-reactivity or nonspecific antibody recognition. We hypothesized that MS could be an ideal tool to address this question. We applied shotgun proteomics after gel fractionation, followed by PRM to further confirm the presence and to quantify the selected non-human peptides. We could reproducibly quantify nine non-human peptides, which mainly originated from bovine milk proteins. In Chapter 4, the human milk glycoproteome is charted. After automated hydrophilic-interaction chromatography (HILIC)-based enrichment of N-glycopeptides, multiple MS methods were performed in Fusion Lumo, allowing identification by higher-energy collisional dissociation product-dependent electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD), quantification by simultaneously scheduled single ion monitoring (SIM) and PRM. We were able to map and quantify one of the most complete N-glycosylation inventories to date in human milk and reveal unique site-specific dynamic changes with a few general trends. In Chapter 5, we focused on a simple albeit ignored parameter improvement in glycopeptide analysis. We compared a set mass range from 2000 to 4000 for tandem mass fragmentation spectra during EThcD and were able to detect several additional c and z ions carrying intact glycans. These fragment ions significantly increased the sequence coverage and, confidence in the identification of the glycopeptides. The use of an extended mass range could benefit the analysis of various classes of glycopeptides. In Chapter 6, we expanded the peptide-centric approach to a protein-centric approach for characterizing the proteoforms of complement component C8 comprehensively. A detailed specification of co-occurring C8 proteoforms was obtained. Within these PTMs, two new N-glycosylation sites and one new O-glycosylation site in C8γ were experimentally observed for the first time. The stoichiometry of all C-mannosylation sites was determined in the various thrombospondin-like (TSP) domains present in C8α and C8β. We proposed that some of these newly identified modifications may play a role in the membrane attack complex formation