LC-MS/MS isomeric profiling of permethylated N-glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients

Abstract

Early stage detection and cancer treatment demand the identification of reliable biomarkers. Over the past decades, efforts have been devoted to assess the variation of glycosylation level as well as the glycan structures of proteins in blood or serum, associated with the development and/or progression of several cancers, including liver. Herein, an LC-MS/MS-based analysis was conducted to define the glycosylation patterns of haptoglobin glycoprotein derived from sera collected from cirrhotic and hepatocellular carcinoma (HCC) patients. The haptoglobin samples were extracted from serum using an antibody-immobilized column prior to the release of N-glycans. A comparison of non-isomeric and isomeric permethylated glycan forms was achieved using C18 and porous graphitic carbon (PGC) columns, respectively. In the case of C18-LC-MS/MS analysis, 25 glycan structures were identified of which 10 sialylated structures were found to be statistically significant between the two cohorts. Also, 8 out of 34 glycan structures identified by PGC-LC-MS/MS were found to be statistically significant, suggesting that isomeric distributions of a particular glycan composition were different in abundances between the two cohorts. The glycan isoform patterns distinguished early stage HCC from cirrhotic patients. Both retention times and tandem mass spectra were utilized to determine the specific isomeric glycan structures. All of the glycan isomers, which were statistically significant, were either branch fucosylated or composed of α-2,6 linked sialic acid moieties. The result of this study demonstrates the potential importance of isomeric separation for defining disease prompted aberrant glycan changes. The levels of several glycan isoforms effectively distinguished early stage HCC from cirrhosis.

Keywords: Cirrhosis; Glycans biomarker discovery; Hepatocellular carcinoma; Isomeric separation; LC-MS/MS; Permethylated glycans.

Figure 1

Unsupervised PCA plot of the glycans that were quantitatively determined byC18-LC-MS/MS analysis. The corresponding loading plot is shown in Figure S1.

Figure 2

Base peak intensity chromatograms of LC-MS/MS analyses of permethylated glycans derived from sera collected from cirrhotic (a) and hepatocellular carcinoma (b) patients. The insets depict the MS of glycans depicting statistically significant differences between the two cohorts. Symbols: as in Scheme 1.

Figure 3

Box plots of normalized abundances of significant glycan structures based on the C18-LC-MS/MS analysis. Y-axis associated with glycans present at low abundances were 10x magnified. Asterisks indicate the level of statistical significance. Boxes depicted 25% and 75% of the samples while the whiskers showed standard error. Symbols: as in Scheme 1.

Figure 4

Heat map of the C18-LC-MS/MS analysis of glycans that have illustrated significant differences between the two cohorts.

Figure 5

Unsupervised PCA plot of the glycans that were quantitatively determined by PGC-LC-MS/MS analysis. The corresponding loading plot was shown in Figure S4.

Figure 6

Box plot of normalized abundances of the glycan structures that depicted significant different among the two cohorts upon PGC-LC-MS/MS analysis, Y-axis associated with glycans present at low abundances were 10x magnified. The significant [5-6-1-3] refers to one of two isomers found with larger retention time, while for [5-6-2-3], to the left side is the first eluted isomer and on the right is the second isomer. Boxes depicted 25% and 75% of the samples while the whiskers showed standard error. Asterisks indicate the level of statistical significance. Symbols: as in Scheme 1.

Figure 7

EIC of biantennary monosialylated branch-fucosylated glycan linkage isomers derived from (a) cirrhotic and (b) hepatocellular carcinoma patients. (c) MS/MS interpretation of biantennary monosialylated branch-fucosylated glycan. Symbols: as in Scheme 1.

Figure 8

Heat map of the PGC LC-MS/MS analysis of glycans that have illustrated significance differences between the two cohorts, where [4-5-0-1,26] refers to the α-2,6 linked sialic acid and [4-5-1-1,26B] refers toα-2,6 linked sialic acid and a branch fucose.

Scheme 1

An example of glycan structure nomenclature used in this report. The model glycan structure contains 4 HexNAc, 3 Mannose, 2 Galactose, 1 Fucose and 1 Neuraminic acid residues. Hence, the nomenclature is [4-5-1-1]. Symbols: , GlcNAc; , mannose; galactose; , fucose; , N-acetylneuraminic acid.