Higher Energy Collisional Dissociation Mass Spectrometry of Sulfated O-Linked Oligosaccharides

Abstract

Sulfation is the final decoration of mucin-type O-linked oligosaccharides before mucins are released into the lumen of the gastrointestinal, respiratory, and genital tracts. Because only a fraction of oligosaccharides undergo this type of modifications in the Golgi apparatus, sometimes also only by dedicated cells, the glycobiology of these low abundant sulfated oligosaccharides is often overlooked. At the same time, the technology to consistently identify and characterize them has been lagging. We adopted higher energy collisional dissociation to characterize sulfated oligosaccharides from porcine gastric and human salivary MUC5B mucins. With this approach we could generate conclusive spectra up to nonasaccharides. Both singly and doubly sulfated oligosaccharides were characterized. By comparing the fragmentation of low-mass fragments of m/ z 100-320 with standards for six-linked and three-linked sulfate, it could be shown that characteristic fragmentation exists, verifying that porcine gastric mucin contains mostly six-linked sulfate to GlcNAc, whereas human MUC5B contains mostly three-linked Gal. When performing ion-trap MS fragmentation, these low-molecular-mass fragments are usually not detected. Hence it can be concluded that to be able to address biological questions of sulfation low-mass fragments are important for the assignment of sulfate position.

Lactose and Bovine Milk Oligosaccharides Synergistically Stimulate B. longum subsp. longum Growth in a Simplified Model of the Infant Gut Microbiome

“…In addition, we applied NMR metabolomics to obtain an untargeted and wide ranging metabolite profile covering several metabolite classes. 48 However, MS-based methods might have provided higher resolution and a better identification of oligosaccharides with higher DP, 49,50 which are very difficult to precisely identify using 1 H NMR. 37,50 In conclusion, coculture and cross-feeding experiments involving P. distasonis, C. perfringens, and B. longum subsp.…”

“…48 However, MS-based methods might have provided higher resolution and a better identification of oligosaccharides with higher DP, 49,50 which are very difficult to precisely identify using 1 H NMR. 37,50 In conclusion, coculture and cross-feeding experiments involving P. distasonis, C. perfringens, and B. longum subsp. longum can contribute to a better understanding of the specific interactions that occur under different prebiotic treatments and add to the discussion of what specific mechanisms are related to the inhibition of C. perfringens in the BMO treatments.…”

Mass Spectrometry-Based Techniques to Elucidate the Sugar Code

“…This, for example, enabled the identification of O -glycans from fungi that are exclusively composed of hexose and hexuronic acid . Likewise, low-mass HCD fragments were recently shown to be diagnostic to the regiochemistry of sulfate modification (3 vs 6) in mucin O -glycans …”

“…403 Likewise, low-mass HCD fragments were recently shown to be diagnostic to the regiochemistry of sulfate modification (3 vs 6) in mucin O-glycans. 404…”

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

“…The MALDI–MS/MS was further extended to more comprehensive and targeted LC–MS/MS analysis, resulting in rapid selection and sequencing of sulfated glycans at high resolution ( 9 , 10 , 11 , 12 , 13 ). Recently, a higher-energy collisional dissociation (HCD) fragmentation technique was used to identify sulfated O-glycans on the mucins5B providing confident identification of larger sulfated glycans ( 14 ). A number of sulfated O-glycans were identified in the mouse secondary lymph nodes by MALDI–MS mapping of fractionated permethylated O-glycans ( 7 ).…”