Journal
TALANTA
Volume 224, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2020.121811
Keywords
Human milk N-Glycoproteomics; Nano LC/Orbitrap MS; Electron-transfer/higher-energy collision dissociation; Ethanol precipitation; Hydrophilic interaction chromatography; Solid phase extraction
Categories
Funding
- K99/R00 Pathway to Independence Career Award, Eunice Kennedy Shriver Institute of Child Health & Development of the National Institutes of Health [R00HD079561]
- Gerber Foundation [2017-1586]
- USDA National Institute of Food and Agriculture [2018-67017-27521]
- National Institute of Health [1S10OD020111-01]
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This study systematically analyzed techniques for profiling N-glycans in human milk and found that a combination of EtOH precipitation, HILIC-SPE and EThcD-fragmentation was the most effective for N-glycopeptide profiling, significantly increasing the number of identified N-glycopeptides and precursor N-glycoproteins. This advancement in methods provides a key step for better understanding the function of glycoproteins in breast milk-fed infants.
Human milk contains numerous N-glycoproteins with functions that provide protection to the infant. Increasing understanding of the functional role of human milk glycoproteins within the infant requires toolsets to comprehensively profile their site-specific glycosylation patterns. However, optimized methods for site-specific glycosylation analysis across the entire human milk proteome are not available. Therefore, we performed a systematic analysis of techniques for profiling the sites and compositions of N-glycans in human milk using liquid chromatography/mass spectrometry. To decrease interference from non-target molecules, we compared techniques for protein extraction, including ethanol (EtOH) precipitation, trichloroacetic acid precipitation, molecular weight cut-off filtration and techniques for tryptic glycopeptide enrichment, including C18-, porous graphitized carbon and hydrophilic interaction liquid chromatography (HILIC)-solid phase extraction (SPE) and acetone precipitation. We compared the capacity of higher-energy collision dissociation, electron-transfer dissociation and electron-transfer/higher-energy collision dissociation (EThcD) to produce fragment ions that would enable effective identification of the glycan composition, peptide sequence and glycosylation site. Of these methods, a combination of EtOH precipitation, HILIC-SPE and EThcD-fragmentation was the most effective for human milk N-glycopeptide profiling. This optimized approach significantly increased the number of N-glycopeptides and precursor N-glycoproteins (246 N-glycopeptides from 29 glycoproteins) compared with a more common extraction approach with no protein extraction and C18 clean-up (62 N-glycopeptides from 11 glycoproteins). The advancement in methods for human milk N-glycoproteins provided by this study represents a key step for better understanding the function of glycoproteins within the breast milk-fed infant.
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