Label-free absolute quantitation of oligosaccharides using multiple reaction monitoring

Qiuting Hong, L Renee Ruhaak, Sarah M Totten, Jennifer T Smilowitz, J Bruce German, Carlito B Lebrilla, Qiuting Hong, L Renee Ruhaak, Sarah M Totten, Jennifer T Smilowitz, J Bruce German, Carlito B Lebrilla

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.

Figures

Figure 1
Figure 1
Response of the method to LNT. (a) The manufacturer default conditions for peptides produced a large amount of unintended LNT fragments (m/z 366) in the MS scan mode. (b) After the instrument optimization, the fragment ion signal was diminished and the quasimolecular ion was increased: blue squares, GlcNAc (N-acetylglucosamine); blue circles, Glc (glucose); yellow circles, Gal (galactose).
Figure 2
Figure 2
Optimization of collision conditions with LNT. (a) The drying gas and sheath gas were maintained at the same temperature. The optimal temperature at 150 °C provides the strongest quasimolecular ion signal. (b) Fragmentor voltage of 250 V provides the lowest degree of fragmentation and the strongest quasimolecular ion signal; 250 V is the minimum setting in this instrument. (c) The rf amplitude of the low-pressure ion funnel at 60 V provides the strongest quasimolecular ion signal. (d) The rf amplitude of the high ion funnel at 100 V provides the lowest degree of fragmentation. The optimum settings are thus 100 and 60 V for the high-pressure and low-pressure ion funnels, respectively. Red triangle: the symbol corresponds to the ratio of the quasimolecular ion (m/z 708) counts to the total ion counts. Blue diamond: the symbol corresponds to the ratio of the fragment ion (m/z 366) counts to the quasimolecular ion (m/z 708) counts. Instrument optimization of the low-pressure ion funnel was performed with rf amplitude at 40 (black triangles), 60 (green circles), and 80 V (red squares), respectively.
Figure 3
Figure 3
Extracted MRM chromatogram. The MRM transitions monitored are provided in Supporting Information Table S-1. Peaks are labeled with corresponding structures. Structures in parentheses mean they could not be specifically resolved or identified. (a) The total dynamic MRM chromatogram monitored for a pooled human milk oligosaccharide sample. Nine abundant HMO compounds can be readily identified and annotated. (b) Lower abundant HMOs are identified and annotated: blue circles, Glc (glucose); yellow circles, Gal (galactose); blue squares, GlcNAc (N-acetylglucosamine); yellow squares, GalNAc (N-acetylgalactosamine); red triangles, Fuc (fucose); purple diamonds, Neu5Ac (N-acetylneuraminic acid).
Figure 4
Figure 4
Box-and-whisker diagrams for secretor mothers (S) and nonsecretor mothers (N) reveal that nonsecretor mothers produce lower amounts of 2′-FL (P < 0.001) and LNFP-I (P = 0.004) than secretor mothers. However, there are some outliers who still produce similar amounts of 2′-FL or/and LNFP-I with secretor mothers, while their total α(1–2) fucosylation is still at relatively low level. The total HMO content produced by nonsecretor mothers is also lower (P = 0.014, Mann–Whitney–Wilcoxon test) than that of secretor mothers using the nonparametric test. (a) 2′-FL. (b) LNFP-I. (c) LNT. (d) LNH. (e) 6′-SL. (f) 3′-SL. (g) LSTc. (h) Total HMO content. Reported P-values are from one-tail Student t test unless specified.

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