Development of a hydrophilic interaction liquid chromatography coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging platform for N-glycan relative quantitation using stable-isotope labeled hydrazide reagents

Abstract

In this work, the capability of newly developed hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for quantitative analysis of N-glycans has been demonstrated. As a proof-of-principle experiment, heavy and light stable-isotope labeled hydrazide reagents labeled maltodextrin ladder were used to demonstrate the feasibility of the HILIC-MALDI-MSI platform for reliable quantitative analysis of N-glycans. MALDI-MSI analysis by an Orbitrap mass spectrometer enabled high-resolution and high-sensitivity detection of N-glycans eluted from HILIC column, allowing the re-construction of LC chromatograms as well as accurate mass measurements for structural inference. MALDI-MSI analysis of the collected LC traces showed that the chromatographic resolution was preserved. The N-glycans released from human serum was used to demonstrate the utility of this novel platform in quantitative analysis of N-glycans from a complex sample. Benefiting from the minimized ion suppression provided by HILIC separation, comparison between MALDI-MS and the newly developed platform HILIC-MALDI-MSI revealed that HILIC-MALDI-MSI provided higher N-glycan coverage as well as better quantitation accuracy in the quantitative analysis of N-glycans released from human serum. Graphical abstract Reconstructed chromatograms based on HILIC-MALDI-MSI results of heavy and light labeled maltodextrin enabling quantitative glycan analysis.

Keywords: Glycomics; HILIC; Hydrazide reagents; MALDI imaging; N-Glycans; Quantitation.

Fig. 1

(A) Overall workflow for quantitative analysis of N-glycans using duplex HDEAT labeling reagents and LC-MALDI-MSI platform. Light and heavy labeled samples were mixed together and separated by HILIC; HILIC traces were collected on MALDI plate and subjected to MSI analysis; the intensity of the analyte peak was extracted from the imaging area and used for quantitation. (B) The chemical structures of light and heavy version of HDEAT. (C) Labeling reaction, hydrazide group of labeling reagent react with aldehyde group in glycans to form stable hydrazone product

Fig. 2

(A) A plot of theoretical log₂(H/L) vs. experimental log₂(H/L) for the labeling ratios at 5:1, 4:1, 3:1, 2:1, 1:1 and reverse labeling at ratios of 1:2, 1:3, 1:4, 1:5 using maltooctose as standards. A weighted linear least squares regression is plotted for the data. (B) The mass spectra of heavy and light labeled maltooctose at the ratios of 5:1 and 1:5, respectively; the ion peak shown here is in protonated form. Note: The experiments were conducted using MALDI-MS

Fig. 3

(A) Mass spectrum of heavy and light labeled (ratio 1:1) N-glycans released from RNase B (pink shaded peaks are light labeled and blue shaded peaks are heavy isotope labeled). (B) Experimental ratios of heavy and light labeled RNase B N-glycans. The error bars stand for standard deviations in three technical replicates. (C) Labeling efficiency comparison of the heavy and light labeled RNase B N-glycans in the two parallel reaction vials. (H hexose, N N-acetylhexoseamine). Note: The experiments were conducted using MALDI-MS

Fig. 4

(A) Re-constructed extracted ion chromatograms of heavy and light labeled maltodextrin labeled at 1:1 ratio based on MSI results. (B) HILIC traces base peak images for heavy and light labeled maltodextrin labeled at 1:1 ratio. Note: The image area of heavy and light labeled maltodextrin overlapped with each other

Fig. 5

(A) Extracted ion images of heavy and light labeled maltodextrin₍₄₎ at 1:1 ratio based on MSI results. (B) Reconstructed LC peak of heavy and light labeled maltodextrin (DP = 4) based on HILIC-MALDI-MSI results. (C) Mass spectrum of heavy and light labeled maltodextrin (DP = 4) in protonated form

Fig. 6

HILIC traces base peak images for heavy and light labeled N-glycans released from human serum and extracted ion imaging results of four representative light labeled N-glycans. All the ions are in sodiated form. Note: Heavy and light labeled N-glycans overlapped with each other; the extracted images presented here are light labeled N-glycans

Fig. 7

(A) Quantitation accuracy and N-glycan coverage comparison between HILIC-MALDI-MSI and MALDI-MS using 1:1 labeled N-glycans released from human serum. The pink shaded part represents the N-glycans only identified and quantified on the HILIC-MALDI-MSI platform. Quantitation accuracy was represented as the percentage difference between experimental ratio and theoretical ratio. The error bar stands for standard deviations of measured ratios from three experimental replicates. (B) Average intensity difference between these two platforms