Mass spectrometric study of N-glycans from serum of woodchucks with liver cancer

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Journal titleRapid Communications in Mass Spectrometry
Pages29832995; # of pages: 13
AbstractWoodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N-glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures – reversed-phase high-performance liquid chromatography (RP-HPLC) and solid-phase extraction (SPE) on a cation-exchange/reversed-phase STRATA-XC cartridge – were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N-glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N-glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N-glycans from total serum in the progression of liver disease. Copyright © 2009 John Wiley & Sons, Ltd. Hepatocellular carcinoma (HCC) is the most common malignancy of the liver and a common cause of cancer-related deaths worldwide.1 Most cases of HCC are secondary to hepatitis B or C viral infection.2 The disease progression is monitored by physical examination of the patient, ultrasound imaging of the liver, and analysis of the patient's serum for a panel of markers.3 The diagnosis of HCC is assisted by the serologic measurement of alpha fetoprotein (AFP) but the lack of sensitivity in detection leads to frequent identification of the disease at an advanced stage. Many molecular changes accompany the development of HCC and could serve as disease markers.4–7 The development of glycoproteomic markers, in particular, is an emerging field with growing importance for the early detection of HCC.8–11 Protein glycosylation is a major post-translational modification with profound biological implications.12–16 Glycosylation of proteins has been shown to change during malignant transformation;17–21 the increase in 1,6-fucosylation is the most notable change reported to accompany the development of HCC.22–24 It was also reported that an alpha(1,3)-fucosylated triantennary glycan was more abundant in the sera of patients with HCC than in patients with cirrhosis and healthy blood donors, whereas the level of a bisecting core alpha(1,6)-fucosylated biantennary glycan was elevated in sera of patients with cirrhosis.25 Enzymatic or chemical release of oligosaccharides from glycoproteins allows structural characterization and quantification.26, 27 Various methods have been employed for the purification and detection of the released glycans.28–31 Mass spectrometry (MS) is currently the most popular technique used for the screening of oligosaccharides in biological samples.32–35 Modern mass spectrometric methods allow highly sensitive characterization of glycans despite the structural diversity of carbohydrates including anomericity, linkage position or branching pattern of sugar moieties.36, 37 The advances in instrumentation and analytical methods facilitated the recently reported analyses of the N-glycome in patients with a variety of diseases.38–43 We have previously shown that phenylhydrazine (PHN) derivatization of oligosaccharides in combination with matrix-assisted laser desorption/ionization (MALDI)-MS allows an efficient characterization of N-glycans in the serum of mice with cancer.44 More than 40 glycans were detected with compositions characteristic of high-mannose, hybrid and complex structures. The goal of the study presented here was to perform a detail characterization of the N-glycans detached from the total serum of woodchucks with and without HCC. Woodchuck is a relevant animal model of viral hepatitis and precisely mimics tumor development seen in humans chronically infected with hepatitis B.24 Detail analyses of glycans from total serum of woodchucks can be useful in the more comprehensive examination of changes in the glycosylation of proteins observed during the development of HCC. In this work, N-glycans were isolated from serum of woodchucks using complementary enzymatic procedures combined with reversed-phase high-performance liquid chromatography (RP-HPLC) or solid-phase extraction (SPE) on cation-exchange/reversed-phase STRATA-XC cartridges. Oligosaccharide pools were derivatized with phenylhydrazine or by permethylation and analyzed by MALDI-QqTOF mass spectrometry.
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AffiliationNRC Institute for Biodiagnostics; National Research Council Canada
Peer reviewedYes
NPARC number19966914
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Record identifier19e5fc7f-f10f-42fc-98d7-09c610404614
Record created2012-05-16
Record modified2016-05-09
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