Abstract
Sample collection, handling and storage are the most critical steps for ensuring the highest preservation of specimens. Pre-analytical variability can influence the results as protein signatures alter rapidly after tissue excision or during long-term storage. Hence, we evaluated current state-of-the-art biobank preservation methods from a glycomics perspective and analyzed O-glycan alterations occurring in the gastric cancer tissues. Paired tumor and adjacent normal tissue samples were obtained from six patients undergoing gastric cancer surgery. Collected samples (n = 24) were either snap-frozen or heat stabilized and then homogenized. Glycans were released from extracted glycoproteins and analyzed by LC-MS/MS. In total, the relative abundance of 83 O-glycans and 17 derived structural features were used for comparison. There was no statistically significant difference found in variables between snap frozen and heat-stabilized samples, which indicated the two preservation methods were comparable. The data also showed significant changes between normal and cancerous tissue. In addition to a shift from high sialylation in the cancer area towards blood group ABO in the normal area, we also detected that the LacdiNAc epitope (N,N’-diacetyllactosamine) was significantly decreased in cancer samples. The O-glycan alterations that are presented here may provide predictive power for the detection and prognosis of gastric cancer.
Highlights
Glycomics is an emerging scientific discipline that seeks to understand the structure and function of glycans in biological systems
We examined the effect of snap freezing versus heat-stabilization technology on gastric O-glycans and O-glycan alterations occurring in the gastric cancer tissues in comparison to adjacent normal mucosa
Aiming to select the best conditions for efficient extraction of proteins from tissue samples, we evaluated a) manual vs. semi-automatic homogenization b) solubilisation buffers with two different detergents – the ionic sodium dodecyl sulfate (SDS) and zwitterionic CHAPS and c) whether sonication was an essential step to solubilize tissue
Summary
Glycomics is an emerging scientific discipline that seeks to understand the structure and function of glycans in biological systems. Freezing, degradation following sample collection or freeze/thaw cycles can influence quality and quantity of proteins/glycoproteins in these matrices. Universal approach for sample preservation involves application of protease inhibitors and cold temperature/freezing. Another preservation technology has been developed combining convective heating under controlled pressure that stop degradation and alterations immediately and permanently[10]. Exploiting the differences in glycan repertoires between cancerous and normal tissues provides opportunities to discover new targets for personalized cancer diagnosis and treatment. Discovery of these cancer-associated modifications of glycans on the glycoproteins may improve on the specificity of existing cancer biomarkers. Adding information on glycan changes to screening of protein levels is an attractive approach
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