Label-Free Absolute Quantitation of Oligosaccharides Using Multiple Reaction Monitoring

Abstract

An absolute quantitation method for measuring free human milk oligosaccharides (HMOs) in milk samples was developed using multiple reaction monitoring (MRM). To obtain the best sensitivity, the instrument conditions were optimized to reduce the source and postsource fragmentation prior to the quadrupole transmission. Fragmentation spectra of HMOs using collision-induced dissociation were studied to obtain the best characteristic fragments. At least two MRM transitions were used to quantify and identify each structure in the same run. The fragment ions corresponded to the production of singly charged mono-, di-, and trisaccharide fragments. The sensitivity and accuracy of the quantitation using MRM were determined, with the detection limit in the femtomole level and the calibration range spanning over 5 orders of magnitude. Seven commercial HMO standards were used to create calibration curves and were used to determine a universal response for all HMOs. The universal response factor was used to estimate absolute amounts of other structures and the total oligosaccharide content in milk. The quantitation method was applied to 20 human milk samples to determine the variations in HMO concentrations from women classified as secretors and nonsecretors, a phenotype that can be identified by the concentration of 2′-fucosylation in their milk.

Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation

“…HMO, human milk oligosaccharide; LDFT, lacto-difucotetraose; LNFP I, lacto-N-fucopentaose I; Resp., response; tetra-iso-LNO, tetrafucosyl-iso-lacto-N-octaose; TFLNH, trifucosyllacto-N-hexaose; 2#FL, 2#-fucosyllactose. (34) for oligosaccharide analysis were used in this study, with some modifications. The capillary voltage and the fragmentor voltage were set at 1800 and 280 V, respectively.…”

“…At least 31 compositions were monitored, representing >100 HMO structures based on our previously published libraries (35,36). For most compositions, one transition was chosen based on the transitions published by Hong et al (34), with some modifications (Supplemental Table 1). …”

“…It also has been used for the analysis of free milk oligosaccharides in bovine milk (32) and rat serum (33); however, the application of MRM to HMOs remains limited. Recently, our laboratory developed an absolute quantitation method for measuring HMOs in milk samples using MRM (34). The individual and total concentrations of chemically reduced HMOs were estimated through the use of a universal response factor obtained from 7 commercial HMO standards.…”

Milk Glycans and Their Interaction with the Infant-Gut Microbiota

“…HMOs are an abundant component of human milk (Bode et al 2016, Petherick 2010), with approximately 5–20 g/L of unbound oligosaccharides present in mature milk (Bode 2006, Hong et al 2014, McGuire et al 2017). Surveys of different human milks have revealed tremendous structural diversity, with intricate molecular structures among and within HMO pools (Kunz et al 2000; Wu et al 2010, 2011).…”

Applications of Multiple Reaction Monitoring to Clinical Glycomics

“…Seven standard HMO (2′-FL, LNT, LNFP-I, 3′-SL, 6′-SL, and LSTc) were used for absolute quantitation and the LOD for each of these compounds was found to be in the low femtomole to attomole range. Using this method, significant differences were observed in the HMO composition of secretors (mothers that can excrete HMO with α1-2 linked fucose) and non-secretors (mothers that do not produce HMO with α1-2 linked fucose) [44] as is illustrated in Figure 3.…”