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

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.