Annotation and structural analysis of sialylated human milk oligosaccharides - PubMed (original) (raw)

. 2011 Feb 4;10(2):856-68.

doi: 10.1021/pr101006u. Epub 2011 Jan 6.

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Annotation and structural analysis of sialylated human milk oligosaccharides

Shuai Wu et al. J Proteome Res. 2011.

Abstract

Sialylated human milk oligosaccharides (SHMOs) are important components of human milk oligosaccharides. Sialic acids are typically found on the nonreducing end and are known binding sites for pathogens and aid in neonates' brain development. Due to their negative charge and hydrophilic nature, they also help modulate cell-cell interactions. It has also been shown that sialic acids are involved in regulating the immune response and aid in brain development. In this study, the enriched SHMOs from pooled milk sample were analyzed by HPLC-Chip/QTOF MS. The instrument employs a microchip-based nano-LC column packed with porous graphitized carbon (PGC) to provide excellent isomer separation for SHMOs with highly reproducible retention time. The precursor ions were further examined with collision-induced dissociation (CID). By applying the proper collision energy, isomers can be readily differentiated by diagnostic peaks and characteristic fragmentation patterns. A set of 30 SHMO structures with retention times, accurate masses, and MS/MS spectra was deduced and incorporated into an HMO library. When combined with previously determined neutral components, a library with over 70 structures is obtained allowing high-throughput oligosaccharide structure identification.

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Figures

Figure 1

Figure 1

(a) Base peak chromatogram (BPC) of enriched sialylated human milk oligosaccharides (SHMOs). (b) Extracted ion chromatogram (EIC) of isomers with neutral mass 1511.6 (with MS inset).

Figure 2

Figure 2

FS-LNH isomers in human milk. The isomers were separated into different fractions using standard HPLC. The selected fractions were then analyzed by Chip/TOF MS (a–g). The structures (inset) were determined as described in the text.

Figure 3

Figure 3

(a) MS/MS of a commercial standard FS-LNH from singly-charged precursor ion (m/z 1512.6). (b) MS/MS of doubly-charged precursor ion (m/z 756.8). (c) MS/MS of FS-LNH obtained from human milk by HPLC fractionation.

Figure 4

Figure 4

Three isomers - 4121a, FS-LNH I, and FS-LNH II, were differentiated by MS/MS under the identical collision energy. Diagnostic peaks (circled) belong only to the specific isomer.

Figure 5

Figure 5

MS/MS spectra serve to distinguish the two isomers – 4121b and FS-LNH III, with nearly identical retention time. Different fragmentation pathways elucidate linear from branched core structures.

Figure 6

Figure 6

Exoglycosidase digestion to determine the structure of FS-LNH III found in HPLC fraction 35.

Figure 7

Figure 7

MS/MS spectra of FS-LNH and FS-LNnH I in the negative ion mode. Z type ions can be used to elucidate the different connectivity within the two isomers.

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