Abstract
Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.
Highlights
Human milk oligosaccharides (HMO) are the third most abundant type of component in human milk [1]
We show that the already known oligosaccharide structures can be assigned according to specific diagnostic ions in combination with the HMO biosynthetic rules, and, novel structure types can be proposed de novo
The matrix-assisted laser desorption/ionization (MALDI) spectrum was acquired from the sample without further purification procedures as an initial oligosaccharide mapping experiment
Summary
Human milk oligosaccharides (HMO) are the third most abundant type of component in human milk [1]. In addition to the complex nature of HMO biosynthesis, additional variety in HMO structures can arise from chemical procedures carried out during sample preparation steps or due to biological enzymatic activity To these belongs the chemical procedure of mild acid hydrolysis in order to remove fucose and sialic acid residues [20]. For large molecular weight HMO, which can possess more complex branching sites, as well as multiple fucosylation sites, the structures are predominantly difficult to define, due to isobaric mixtures. This is frequently solved by the application of liquid chromatography (LC) protocols, which allow isomer separation prior to MS analysis [2,3]. The additional advantages of the MALDI Q-TOF MS analysis are the sensitivity, speed, high dynamic range and ability to analyse data in more detail on the same sample spot once the preliminary data analysis is completed
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