Profiling of human serum glycans associated with liver cancer and cirrhosis by IMS-MS

Abstract

Aberrant glycosylation of human glycoproteins is related to various physiological states, including the onset of diseases such as cancer. Consequently, the search for glycans that could be markers of diseases or targets of therapeutic drugs has been intensive. Here, we describe a high-throughput ion mobility spectrometry/mass spectrometry analysis of N-linked glycans from human serum. Distributions of glycans are assigned according to their m/z values, while ion mobility distributions provide information about glycan conformational and isomeric composition. Statistical analysis of data from 22 apparently healthy control patients and 39 individuals with known diseases (20 with cirrhosis of the liver and 19 with liver cancer) shows that ion mobility distributions for individual m/z ions appear to be sufficient to distinguish patients with liver cancer or cirrhosis. Measurements of glycan conformational and isomeric distributions by IMS-MS may provide insight that is valuable for detecting and characterizing disease states.

Figure 1

Schematic diagram of the NanoMate-IMS-TOF instrument.

Figure 2

Two-dimensional t_D(m/z) plot of mobility-dispersed glycan ions originating from human serum. Glycan structures were assigned by comparison of measured m/z values with m/z values cited in the literature. S represents sialic acid, H represents a hexose (galactose or mannose), and N represents N-acetyl glucosamine. In cartoon images, solid squares represent N-acetyl glucosamine, open circles represent mannose, solid circles represent galactose, and diamonds represent sialic acid. Two structural isomers of S₁H₅N₄ have sialic acid added to the antenna either on the α-1,3-linked mannose residues or α-1,6-linked mannose residues.

Figure 3

Overlap of normalized total mass spectra for a set (three samples) of glycans isolated from human serum of a patient with liver cancer (bottom), a healthy patient (middle), and a patient with a liver cirrhosis (top). Glycan structures were assigned by comparison of measured m/z values with m/z values of glycans found in the literature, and the assignment is shown on the top of the plot. S represents sialic acid, H represents hexose (mannose or galactose), N represents N-acetyl glucosamine, and F represents fucose.

Figure 4

(A) PCA of drift time profiles for glycan [S₁H₅N₄ + 3Na]³⁺ (m/z = 826.0). (B) PCA of drift time profiles for glycan [S₂H₅N₄ + 3Na]³⁺ (m/z = 946.7). Both analyses were done a total of 61 samples, that is, 19 glycan samples from serum of patients with liver cancer (HC samples, green circles), 22 glycan samples from healthy patients (NC-samples, red squares), and 21 glycan sample from patients with liver disease cirrhosis (QT-samples, purple triangles).

Figure 5

(A) Probing conformational changes of glycan isomers by IMS-IMS–MS. Precursor and mobility selected drift time distributions for [S₂H₅N₄ + 3Na]³⁺ (m/z = 946.7) at three different selection times (shown by dashed lines). The drift time distributions for mobility selected precursors are obtained after gentle activation of ions at IA2. (B) Probing conformational changes of isomers by IMS-IMS–MS (continued). Precursor and mobility selected drift time distributions for [S₁H₅N₄ + 3Na]³⁺ (m/z = 826.0) at three different selection times (shown by dashed lines). The drift time distributions for mobility selected precursors are obtained after gentle activation of ions at IA2. Selections made for low mobility precursors are dominated by similar features with an identical distribution after ion activation indicating presence of one isomer. High-mobility region is dominated by features that do not show similar distribution upon ion activation indicating presence of another isomer.