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A team of researchers performed quantitation of N-glycans from the human alfa-acid glycoprotein (haGP) that revealed N-glycans as potential biomarkers for pancreatic cancer. hAGP is an N-linked glycoprotein (single polypeptide) with 183 amino acids and molecular weight 41 kDa.
hAGP contains about 45 % of carbohydrate by weight and up to 16 Sialic acid residues in the carbohydrate moieties. It contains five heteropolysaccharides that are linked to the asparaginyl residue of the protein through N-glycosidic linkage. Glycoproteins are those proteins that contain carbohydrate moieties covalently attached to them.
Glycoproteins may be altered in most of the diseases including congenital disorders of glycosylation, chronic inflammations, and cancer. Many of them have been reported as tumor markers such as for instance prostate-specific antigen for prostate cancer. However, some of them are not cancer-specific enough as they can be overexpressed in some other diseases as well.
Patterns of glycosylation such as fucosylation, sialylation or extent, and types of branching of glycans of the glycoproteins have been reported to be altered in glycans structure of the glycoprotein associated with cancer and inflammatory diseases. Therefore, researchers were searching for a specific glycans structure that can serve as a potential biomarker for pancreatic cancer.
N-glycans for potential biomarkers
Previously, glycan reductive isotope labeling (GRIL) using [12C6] and [13C6] coded aniline followed by µZIC-HILIC-ESI-MS analysis was established and optimized for the relative quantitation of the glycans. This strategy permits a reliable comparison between glycosylation patterns of a glycoprotein in the different pathological conditions.
For this purpose, researchers isolated serum hAGP samples from healthy patients (control) and patients with different stages of pancreatic cancer. They purified hAGP using immunoaffinity chromatography and accessed the purity using the Nanodrop spectrophotometer and SDS-PAGE.
After purification of the hAGP, N-glycans were released from the hAGP by treating the sample with 0.5 % β-mercaptoethanol, 0.5 % SDS in ammonium bicarbonate of pH 7.9 along with 1 % (v/v) NP-40 alternatives. β-mercaptoethanol and SDS were added to denature the 3D structure of the protein while 0.5 µl of Peptide-N-glycosidase F (PNGase F) was added to the reaction mixture to release the glycans from the polypeptide chain.
The released N-glycans from hAGP purified those glycans using hypercarb cartridge (solid-phase extraction). After purification, researchers performed Glycan Reductive Isotope Labeling (GRIL) using [12C6] / [13C6] coded aniline during which N-glycans from control and standard hAGP were labeled with [12C6]-Aniline while N-glycans from pathological sample hAGP were labeled with [13C6]-aniline.
After labeling, researchers analyzed those labeled-glycans using the Zwitterionic Hydrophobic Interaction Capillary Liquid Chromatography coupled with Electro Spray Ionization Mass Spectrometry (µZIC-HILIC-ESI-MS). Researchers were able to distinguish between the hAGP from healthy and patients with pancreatic cancer on the basis of relative abundance of certain selected N-glycan species (especially glycans such as 3Ant3Ac1Fuc and 4Ant3NeuAc1Fuc).
µZIC-HILIC-ESI-MS analysis of N-glycans revealed that there is an increase in fucosylated N-glycans and a decrease in non-fucosylated N-glycans in pancreatic cancer as compared to that of the control sample. In conclusion, fucosylated N-glycans: 3Ant3NeuAc1Fuc and 4Ant3NeuAc1Fuc can be used as potential biomarkers for pancreatic cancer.
Reference: Analytica Chimica Acta (Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis)
Article DOI: 10.1016/j.aca.2015.02.008