Bladder Cancer Glycoproteomics Differential Studies Based On FFPE Sections

Protein glycosylation is closely related to various biological functions of proteins.Abnormal protein glycosylation is related to the occurrence of various cancers,which is manifested in the appearance of unique N-linked glycans and N-linked glycoproteins in cancer tissues.Bladder cancer is a malignant tumor that occurs in the bladder mucosa and is one of the most common malignant tumors in the urinary system.There are few studies on the glycan of bladder cancer in clinical cases.,and it is hard to fully understand the role of glycan in bladder cancer.In-depth glycomics analysis not only provides cancer candidate markers for future clinical detection,but also contributes to understanding the mechanism of occurrence and development of bladder cancer.In addition,the glycomics and glycoproteomics that use FFPE tissue sections as research materials are also constantly developing,which gives us the opportunity to perform in-depth glycomics analysis of bladder cancer from FFPE tissue sections.In order to understanding the N-linked glycans in bladder cancer FFPE tissue sections in this study.we developed a method for in situ extraction of N-linked glycans from FFPE tissue sections and mass spectrometry analysis.In total of 16 pairs tumor and peritumoral tissue of bladder cancer were performed in this study.After deparaffinization,rehydration and antigen retrieval of FFPE tissue sections,the glycans were released by PNGase F enzymatic and permethylated.Then the glycans were detected by MALDI-TOF/TOF-MS and the spectra were resolved.The result shows that highmannose-type glycans N2H6,N2H7,N2H8,N2H9 and complex-type glycans N5H6F1 were significantly increased.Meanwhile,high-mannose-type glycan N2H5,hybrid-type glycans N3H5 and complex glycans N3H4,N4H4,N5H6F1S2 were significantly decreased in the bladder cancer tissue.Receiver Operating Characteristic(ROC)curve analysis showed that the biantennary N-linked glycans N3H4 and N4H4,alone or combined,could be used to distinguish tumor tissue and peritumoral tissue of bladder cancer,which may be potential biomarkers of bladder cancer.In order to further reveal the differences of glycoprotein expression in bladder cancer,this study further developed an in situ extraction of N-linked glycoproteins from FFPE tissue sections and revealed the differences of N-linked glycoproteome of bladder cancer.After deparaffinization,rehydration and antigen retrieval of FFPE tissue sections,the peptides were released in situ from the tissue by trypsin.After desalting,TMT-labeled glycopeptides from tumor and paracancerous sections were enriched by the MAX column,and then detected by LC-MS/MS.It showed that a total of 230N-linked glycoproteins were identified,about half of them are located in the extracellular area,extracellular space,etc.The data analysis of up-and down-regulated glycoproteins is consistent with previous research trends;and it is found that glycosylation site specificity exists in proteins closely related to cancer development,such as fibronectin.In conclusion,we established a method for the analysis of glycomics and glycoproteomics based on bladder cancer FFPE tissue sections.This study revealed aberrant N-glycosylation modifications of proteins in bladder cancer tumor tissues,and identified N-linked glycans N3H4 and N4H4 as potential biomarkers for bladder cancer.In addition,N-glycoproteins have been found to have glycosylation site specificity in proteins such as fibronectin that are closely related to cancer development.