Systematic examination of protein extraction, proteolytic glycopeptide enrichment and MS/MS fragmentation techniques for site-specific profiling of human milk N-glycoproteins

TALANTA

卷:224

Article

Kim, Bum Jin¹  Dallas, David C.¹ 

[1] Oregon State Univ, Coll Publ Hlth & Human Sci, Sch Biol & Populat Hlth Sci, Nutr Program, Corvallis, OR 97331 USA

关键词: Human milk N-Glycoproteomics;    Nano LC/Orbitrap MS;    Electron-transfer/higher-energy collision dissociation;    Ethanol precipitation;    Hydrophilic interaction chromatography;    Solid phase extraction;   

DOI  :  10.1016/j.talanta.2020.121811

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

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