Utrecht U Biomass - Milk glycopeptides

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Milk_Glycopeptide.sky.zip2019-05-27 22:12:41523283286,87427
Milk glycopeptide across lactation
Data License: CC BY 4.0 | ProteomeXchange: PXD022415
  • Organism: Homo sapiens
  • Instrument: Orbitrap Fusion Lumos
  • SpikeIn: Yes
  • Keywords: Human milk; Glycoproteomics
  • Lab head: Albert J.R. Heck Submitter: Jing Zhu
Abstract
Protein N-glycosylation is an essential post-translational modification (PTM) frequently occurring on human milk proteins. These PTMs assist in protecting the infant's health and function amongst 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 HILIC-based cartridges enabling the proteome-wide monitoring of N-glycopeptides using just 150 μg of breast milk protein digestion. Reproducibility in enrichment and quantitation is demonstrated in triplicate measurements of milk samples taken along an extended lactation period. Using higher energy c-trap dissociation (HCD) triggered electron-transfer higher-energy collision dissociation (EThcD), we were able to map around 1700 glycopeptides from 110 glycoproteins occurring on 191 glycosites, of which 43 sites were not before reported with experimental evidence. We next quantified many of these glycopeptides using a targeted proteomics approach with scheduled selective ion monitoring (SIM) and parallel reaction monitoring (PRM). In a 90 min gradient, we could quantitatively monitor 318 glycopeptides originating from 51 glycoproteins. Although each glycoprotein, each N-glycosylation site and each glycan attached revealed its own dynamic changes, we did observe a few general trends. Overall fucosylation especially terminal fucosylation increased across the lactation period.
Experiment Description
We first got the identification of milk proteins and their intensities across lactation by shotgun proteomics. We set out to enrich N-glycopeptides from human milk in an automated enrichment workflow using AssayMAP Bravo platform and HILIC-based cartridges. N-glycopeptides from 150 µg of milk serum tryptic digestion were captured and step eluted in technical triplicates, which were then measured in HCD triggered EThcD and searched by Byonic. We then built a targeted assay with scheduled SIM and PRM to quantify the intact glycopeptides, with spiked PRTC peptides to supervise the variability between runs and retention time shifts. Data analysis was performed in Skyline. The intensity of glycopeptide was normalized to the intensity of its protein of origin.
Sample Description
In brief, we first collected milk samples longitudinally from the same individual across early, transitional and mature lactational stages, totaling 9 time-points including weeks 1, 2, 3, 4, 6, 8, 10, 12 and 16. By centrifugation and ultracentrifugation, the separated milk serum was subsequently tryptic digested and the final peptide concentrations were estimated.
Created on 5/27/19, 10:15 PM