Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes

Abstract

Protein N-glycosylation on human milk proteins assists in protecting an infant's health and functions among others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother's milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein-and N-site-specific quantitative information. Here, we describe an automated platform using hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact Nglycopeptides using just a digest of 150 μg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants.

Towards structure-focused glycoproteomics

“…patients with prostate cancer (PCa) and benign prostatic hyperplasia [ 52 ] or over a period of interest, e.g. longitudinal profiling during lactation [ 66 ] or neonatal heart development [ 69 ].…”

“…In the surveyed studies, data were most frequently collected using data-dependent acquisition (DDA) by employing higher-energy collision dissociation (HCD-), stepped collision energy (SCE)-HCD-, electron-transfer dissociation (ETD)- and/or electron-transfer/higher-energy collision dissociation (EThcD-) MS/MS acquisition strategies (discussed below). Our survey also illustrated that targeted re-isolation and orthogonal fragmentation of glycopeptide ions are increasingly performed using diagnostic oxonium ions arising from HCD-MS/MS commonly referred to as product-dependent (pd) acquisition [ 60 , 66 , 67 , 71 ]. Data-independent acquisition (DIA), albeit less common, has also been used to profile mixtures of glycopeptides [ 57 , 72–76 ].…”

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors

“…Although they may overcome potential proteoform biases in antibody-specificity for IP, the increased sample complexity and resulting co-elution of (glyco-)peptides in such approaches introduces significant biases due to ion suppression and undersampling. 39 , 40 …”

Optimization of a human milk–directed quantitative sIgA ELISA method substantiated by mass spectrometry

“…Details of subjects and the human milk samples used in this study as well as label-free quantification (LFQ) of shotgun proteomics methodologies have been extensively described previously [14,15]. Briefly, human milk samples were collected from two individual donors across weeks 1, 2, 3, 4, 6, 8, 10, 12, and 16 of lactation under standardized conditions [16].…”

“…However, it is known that removal of these glycans decreases the interaction of sIgA with gram-positive bacteria [37], indicating that changing the glycosylation of sIgA changes the functionality. Future work on the effects of glycosylation and ELISA development could implement the use of MS as a complementary method as glycan annotation by MS has advanced in recent years, enabling the analysis of both glycan composition and glycosite annotation in human milk [14].…”