Abstract
Human milk oligosaccharides (HMOs) exhibit various biological activities for infants, such as serving as prebiotics, blocking pathogens, and aiding in brain development. HMOs are a complex mixture of hetero-oligosaccharides that are generally highly branched, containing multiple structural isomers and no intrinsic chromophores, presenting a challenge to both their resolution and quantitative detection. While liquid chromatography-mass spectrometry (LC-MS) has become the primary strategy for analysis of various compounds, the very polar and chromophore-free properties of native glycans hinder their separation in LC and ionization in MS. Various labeling approaches have been developed to achieve separation of glycans with higher resolution and greater sensitivity of detection. Here, we compared five commonly used labeling techniques [by 2-aminobenzamide, 2-aminopyridine, 2-aminobenzoic acid (2-AA), 2,6-diaminopyridine, and 1-phenyl-3-methyl-5-pyrazolone] for analyzing HMOs specifically under hydrophilic-interaction chromatography-mass spectrometry (HILIC-MS) conditions. The 2-AA labeling showed the most consistent deprotonated molecular ions, the enhanced sensitivity with the least structural selectivity, and the sequencing-informative tandem MS fragmentation spectra for the widest range of HMOs; therefore, this labeling technique was selected for further optimization under the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) conditions. The combination strategy of 2-AA labeling and PGC-MS techniques provided online decontamination (removal of excess 2-AA, salts, and lactose) and resolute detection of many HMOs, enabling us to characterize the profiles of complicated HMO mixtures comprehensively in a simple protocol.
Highlights
IntroductionHuman milk oligosaccharides (HMOs) have been assigned to a variety of important biological functions for infants (Newburg, 2013; Bode, 2015; Wicinski et al, 2020), such as prevention of pathogens binding to epithelial cell surfaces (Hester et al, 2013; Gonia et al, 2015; Triantis et al, 2018; Ha et al, 2020), functioning as prebiotics (Boehm et al, 2005; Marcobal and Sonnenburg, 2012; Underwood et al, 2015; Walsh et al, 2020), and enhancing brain development (Oliveros et al, 2018; Analytical Strategies for HMOs ProfilingDocq et al, 2020; Wu et al, 2020)
Protocol 1: lactose-free R-Human milk oligosaccharides (HMOs) were derivatized by different labeling techniques, purified by solid-phase extraction (SPE) approaches, and analyzed by the HILIC-mass spectrometry (MS) approach; Protocol 2: lactose-free R-HMOs were derivatized by the optimal labeling technique proposed, purified by SPE approaches, and analyzed by the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) approach; Protocol 3: raw C-HMOs with lactose were derivatized by the optimal labeling technique and analyzed by the porous graphitic carbon chromatography (PGC)-MS approach directly
This study evaluated the influence of glycan labeling, sample preparation, and liquid chromatography-mass spectrometry (LC-MS) methods on the compositional profiling analysis of HMOs
Summary
Human milk oligosaccharides (HMOs) have been assigned to a variety of important biological functions for infants (Newburg, 2013; Bode, 2015; Wicinski et al, 2020), such as prevention of pathogens binding to epithelial cell surfaces (Hester et al, 2013; Gonia et al, 2015; Triantis et al, 2018; Ha et al, 2020), functioning as prebiotics (Boehm et al, 2005; Marcobal and Sonnenburg, 2012; Underwood et al, 2015; Walsh et al, 2020), and enhancing brain development (Oliveros et al, 2018; Analytical Strategies for HMOs ProfilingDocq et al, 2020; Wu et al, 2020). Liquid chromatography (LC) and capillary electrophoresis (CE) coupled with UV adsorption or laser-induced fluorescence (LIF) detection have previously been the major methods for HMO (relatively) quantification analysis by tagging the native oligosaccharides with chromophores and fluorophores (Ruhaak et al, 2010). Even though these methods permit higher UV sensitivity or fluorescence detection, they do not provide specific structural information, and this is a key point due to the highly complex nature of milk glycan mixtures. Future research is needed to explore suitable labeling approaches for HMOs to enhance the analytical performance of LC-MS techniques (Ruhaak et al, 2010)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
