Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards

Abstract

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Keywords: N-glycan; mass spectrometry; permethylation; quantification; standard.

Figure 1

Heterogeneity and mass purity of N-glycan standards. Aliquots of N-glycan standards 107, 108, and 121 were analyzed as 2-AB derivatives by HPLC or as permethylated glycans by MALDI-TOF MS and NSI-LTQ/Orbitrap MS. All three analytic platforms revealed the presence of a major component, corresponding to the expected structure, and minor but structurally related components. (A) Plot of the percent of the total signal contributed by the expected structure across all three platforms (mean ± SD, n = 6). The low magnitude of the error indicates that the detected heterogeneity of the standards reflects true structural diversity in the material and was not generated by the analytic technique. (B) Mass purity for the N-glycan standards calculated by referencing 2-AB fluorescence of N-glycan peaks to known amounts of 2-AB-labeled malto-series standards following HPLC separation or by referencing N-glycan signals to an acetone internal standard by NMR. Mass purity values obtained by fluorescence-coupled HPLC and by NMR were averaged for each of the N-glycan samples (mean ± SD, n = 10).

Figure 2

Linearity of MS responses for N-glycan standards. Known amounts of N-glycan standards 107, 108, and 121 were permethylated and analyzed as a dilution series ranging from 0.5 nM to 6.5 μM at infusion. Regression analysis of all data points from the three standards yields a high coefficient of correlation (r² > 0.95) and linearity over 4 orders of magnitude. Black square, standard 107; red diamond, standard 108; gray triangle, standard 121.

Figure 3

Linearity of MS/MS responses for major N-glycan standard fragments. For each of the N-glycan standards 107, 108, and 121, MS/MS spectra were obtained by NSI-LTQ/Orbitrap mass spectrometry. Signal intensities for the two most abundant fragment ions for each standard were quantified at multiple dilutions of the parent compound. (A) Fragmentation of standard 107 (parent m/z = 946.98, doubly charged) yielded major products at m/z = 835 (black diamonds, loss of 1 HexNAc) and at m/z = 706 (gray circles, loss of 2 HexNAc residues). (B) Fragmentation of standard 108 (parent m/z = 1087.54, triply charged) yielded major products at m/z = 958 (black diamonds, loss of 1 HexNAc) and at m/z = 828 (gray circles, loss of 2 HexNAc residues). (C) Fragmentation of standard 121 (parent m/z = 1407.68, doubly charged) yielded major products at m/z = 1220 (black diamonds, loss of 1 NeuAc, doubly charged) and at m/z = 821 (gray circles, loss of 1 NeuAc, triply charged). For all three N-glycan standards, regression analysis of MS/MS fragment signal intensities as a function of standard concentration at infusion were linear with r² > 0.996.

Figure 4

Linearity of MS responses for malto-series standards. Known amounts of malto-series oligosaccharide standards dp4–7 were permethylated and analyzed as a dilution series ranging from 30 nM to 3 μM at infusion. Regression analysis of all data points from the four malto-series standards yields a high coefficient of correlation (r² > 0.98) and linearity over the assayed range. Black square, dp4; red diamond, dp5; gray triangle, dp6; white circle, dp7.

Figure 5

Molar responses for permethylated N-glycan and malto-series standards are independent of glycan mass. The slopes of the standard curves for N-glycan standards 107, 108, and 121 and malto-series standards dp4–7 were plotted as a function of the mass of the standard. Minimal deviation across the indicated mass range was detected, indicating that permethylation effectively equalizes ionization efficiency across a broad range of glycan structures.

Figure 6

Comparability of signal responses for malto-series standards across three analytic platforms. Malto-series oligosaccharide standards dp3–7 were analyzed as permethylated derivatives by NSI-LTQ/Orbitrap MS ((A) full MS; (B) charge states deconvoluted by Xtract), by MALDI-TOF MS (C), or as 2-AB derivatives by HPLC (D). The malto-series standard mixture was prepared with 10% more dp4 than the other malto-oligosaccharides to readily confirm elution positions and standard identities.

Figure 7

Molar response factors for malto-series standards measured by HPLC fluorescence detection and mass spectrometry. Molar response factors, calculated as detector signal per pM concentration of standard, were measured for the malto-series oligosaccharide standards dp3–7 at a range of concentrations (35 nM to 3 μM). The mean ± SEM of the molar response factors across the analyzed concentrations for each standard is presented for HPLC coupled to fluorescence detection (black squares; n = 9 for each standard) and for NSI-MS (red diamonds; n = 7 for each standard). For the malto-series standards dp4–7, the relationship between molar response and standard mass is identical for HPLC and MS.

Figure 8

Permethylation of malto-series standards with methyl iodide containing stable isotopes of carbon or hydrogen yields equivalent MS responses. The indicated malto-series standards were permethylated with ¹²C-, ¹³C-, or deuterated methyl iodide (¹²C-MeI, ¹³C-MeI, or CD₃I, respectively) and analyzed by NSI-LTQ/Orbitrap MS. Instrument responses for standards permethylated with ¹²C-MeI or CD₃I were normalized to the responses obtained for standards permethylated with ¹³C-MeI, which were set to 100. Normalized responses were averaged for all replicates of the standards permethylated with ¹²C-MeI or CD₃I and are plotted as mean ± SD (n = 3 for each standard). The minor variation around the mean indicates that standards can be permethylated with reagents that induce mass shifts most suitable for the biological sample being studied.

Figure 9

Recovery of MS signal responses for exogenous standards mixed into a biological matrix. The N-glycan standards 107, 108, and 121 were permethylated with ¹³C-MeI and analyzed as a standard mix alone (A). N-Linked glycans prepared from Drosophila melanogaster embryos were permethylated with ¹²C-MeI and analyzed alone (B) or after being supplemented with the ¹³C-MeI permethylated N-glycan standard mix (C). Recovery of signal intensities for standards and sample peaks indicates the lack of suppression or matrix interference for quantification by exogenous standard.

Figure 10

Quantification of mouse brain N-glycans by reference to exogenous standards. N-Linked glycans were released from mouse brain glycoproteins and permethylated with ¹²C-MeI. N-Glycan and malto-series exogenous standards were permethylated with ¹³C-MeI, combined, and spiked into preparations of mouse brain-derived glycans. The eight most abundant mouse brain glycans were quantified by reference to each of the exogenous standards (107, 108, 121, and dp4–7). The calculated amount of each of the brain glycans (mean ± SD) is presented for quantification by reference to malto-series standards (All dp, gray bars; n = 4), N-glycan standards (All N-Gly, red bars; n = 3), or all glycan standards (All Gly, white bars; n = 7). Quantification of brain glycans yielded similar results regardless of which standard set was used.