Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry. The isomeric glycan structures of AZGP1 in these spots were systematically characterized both at composition level and at structure level. Our results revealed 10 glycan compositions for salivary AZGP1, including core fucosylated glycans on Asn128 and sialylated glycans on Asn109 and Asn112. We further compared the glycan structures of salivary AZGP1 from lung cancer group and control group. Accordingly, 14 and 7 potential glycan structures were successfully revealed, respectively. In total, 15 glycan compositions and 22 potential glycan structures were identified and characterized for AZGP1, including some different structures with the same compositions. In particular, 5 potential glycan structures were identified as lung cancer unique signatures. Our developed strategy holds promise for thorough identification of glycan structures on a target glycoprotein biomarker. In-depth characterization of its glycan structures will ultimately enhance its sensitivity and specificity for cancer detection.
Shixian Liu,Yuting Hou,Zirui Li,Chenyu Yang,Guozhen Liu
A fully integrated device for salivary detection with a sample-in-answer-out fashion is critical for noninvasive point-of-care testing (POCT), especially for the screening of contagious disease infection. Microfluidic paper-based analytical devices (μPADs) have demonstrated their huge potential in POCT due to their low cost and easy adaptation with other components. This study developed a generic POCT platform by integrating a centrifugal microfluidic disc with μPADs to realize sample-to-answer salivary diagnostics. Specifically, a custom centrifugal microfluidic disc integrated with μPADs is fabricated, which demonstrated a high efficiency in saliva treatment. To demonstrate the capability of the integrated device for salivary analysis, the SARS-CoV-2 Nucleocapsid (N) protein, a reliable biomarker for SARS-CoV-2 acute infection, is used as the model analyte. By the chemical treatment of the μPAD surface, and by optimizing the protein immobilization conditions, the on-disc μPADs were able to detect the SARS-CoV-2 N protein down to 10 pg mL–1 with a dynamic range of 10–1000 pg mL–1 and an assay time of 8 min. The integrated device was successfully used for the quantification of the N protein of pseudovirus in saliva with high specificity and demonstrated a comparable performance to the commercial paper lateral flow assay test strips.
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