Potential biomarkers identified by tandem mass tags based quantitative proteomics for diagnosis and classification of Guillain-Barré syndrome.

BACKGROUND AND PURPOSE: Guillain-Barré syndrome (GBS) is an acute inflammatory autoimmune and demyelinating disease of the peripheral nervous system. Currently, valid biomarkers are unavailable for the diagnosis of GBS. METHODS: A comparative proteomics analysis was performed on the cerebrospinal fluid (CSF) from 10 patients with GBS and 10 patients with noninflammatory neurological disease (NND) using the tandem mass tags technique. The differentially expressed proteins were analyzed by bioinformatics, and then the candidate proteins were validated by the enzyme-linked immunosorbent assay method in another cohort containing 160 samples (paired CSF and plasma of 40 patients with GBS, CSF of 40 NND patients and plasma of 40 healthy individuals). RESULTS: In all, 298 proteins were successfully identified in the CSF samples, of which 97 differentially expressed proteins were identified in the GBS and NND groups. Three key molecules were identified as candidate molecules for further validation. The CSF levels of TGOLN2 and NCAM1 decreased in GBS patients compared with NND patients, whereas the CSF levels of APOC3 increased. The enzyme-linked immunosorbent assay results were consistent with our proteomics analysis. Interestingly, in the validation cohort, serum APOC3 levels in the GBS group were consistent with those in the CSF samples and significantly higher than those in the healthy control group. CONCLUSIONS: Our preliminary data suggest that the CSF protein expression profile of patients with GBS is different from that of patients with NND. Moreover, alterations of TGOLN2, NCAM1and APOC3 may be used as novel biomarkers for identifying patients with GBS.

Dual-detection fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike RBD-ACE2 blocking neutralizing antibody.

The global effort against the COVID-19 pandemic dictates that routine quantitative detection of SARS-CoV-2 neutralizing antibodies is vital for assessing immunity following periodic revaccination against new viral variants. Here, we report a dual-detection fluorescent immunochromatographic assay (DFIA), with a built-in self-calibration process, that enables rapid quantitative detection of neutralizing antibodies that block binding between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2). Thus, this assay is based on the inhibition of binding between ACE2 and the RBD of the SARS-CoV-2 spike protein by neutralizing antibodies, and the affinity of anti-human immunoglobulins for these neutralizing antibodies. Our self-calibrating DFIA shows improved precision and sensitivity with a wider dynamic linear range, due to the incorporation of a ratiometric algorithm of two-reverse linkage signals responding to an analyte. This was evident by the fact that no positive results (0/14) were observed in verified negative samples, while 22 positives were detected in 23 samples from verified convalescent plasma. A comparative analysis of the ability to detect neutralizing antibodies in 266 clinical serum samples including those from vaccine recipients, indicated that the overall percent agreement between DFIA and the commercial ELISA kit was 90.98%. Thus, the proposed DFIA provides a more reliable and accurate rapid test for detecting SARS-CoV-2 infections and vaccinations in the community. Therefore, the DFIA based strategy for detecting biomarkers, which uses a ratiometric algorithm based on affinity and inhibition reactions, may be applied to improve the performance of immunochromatographic assays.

Discovery of Novel Biomarkers for Diagnosing and Predicting the Progression of Multiple Sclerosis Using TMT-Based Quantitative Proteomics.

Objective: Here, we aimed to identify protein biomarkers that could rapidly and accurately diagnose multiple sclerosis (MS) using a highly sensitive proteomic immunoassay. Methods: Tandem mass tag (TMT) quantitative proteomic analysis was performed to determine the differentially expressed proteins (DEPs) in cerebrospinal fluid (CSF) samples collected from 10 patients with MS and 10 non-inflammatory neurological controls (NINCs). The DEPs were analyzed using bioinformatics tools, and the candidate proteins were validated using the ELISA method in another cohort comprising 160 samples (paired CSF and plasma of 40 patients with MS, CSF of 40 NINCs, and plasma of 40 healthy individuals). Receiver operating characteristic (ROC) curves were used to determine the diagnostic potential of this method. Results: Compared to NINCs, we identified 83 CSF-specific DEPs out of a total of 343 proteins in MS patients. Gene ontology (GO) enrichment analysis revealed that these DEPs are mainly involved in platelet degranulation, negative regulation of proteolysis, and post-translational protein modification. Pathway enrichment analysis revealed that the complement and coagulation cascades, Ras signaling pathway, and PI3K-Akt signaling pathway are the main components. Insulin-like growth factor-binding protein 7 (IGFBP7), insulin-like growth factor 2 (IGF2), and somatostatin (SST) were identified as the potential proteins with high scores, degree, and centrality in the protein-protein interaction (PPI) network. We validated the expression of these three proteins using ELISA. Compared to NINCs, the level of CSF IGFBP7 was significantly upregulated, and the level of CSF SST was significantly downregulated in the MS group. Conclusion: Our results suggest that SST and IGFBP7 might be associated with the pathogenesis of MS and would be helpful in diagnosing MS. Since IGFBP7 was used to classify relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients, therefore, it may act as a potential key marker and therapeutic target in MS.

EMT-Related Markers in Serum Exosomes are Potential Diagnostic Biomarkers for Invasive Pituitary Adenomas.

PURPOSE: Assessing the invasiveness of pituitary adenomas (PAs) is critical to making the best surgical and treatment plan. However, it is difficult to determine the invasiveness of pituitary adenomas based on current clinical methods, such as imaging and histological methods. The present article aims to investigate noninvasive methods to discover viable biomarkers for invasive pituitary adenomas and provide a basis for early intervention of pituitary adenomas. METHODS: E-cadherin, N-cadherin, Epcam, TGF-ß, Smad3, and Smad7 were detected in the tissues and exosomes in 10 cases of invasive PAs and 10 cases of noninvasive PAs by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemical analysis. RESULTS: Compared with that in the noninvasive group, the expression of N-cad in the exosomes of the invasive group was significantly increased, and the expression of E-cad and Epcam was reduced. In the invasive group, the expression levels of TGF-ß1 and Smad3 were reduced. These results were consistent across exosomes and groups. In further cell experiments, the EMT ratio in the SIS3 treatment group, and especially in the TGF-ß1 plus SIS3 treatment group (P <0.001), was significantly increased, and the EMT ratio was significantly lower when one-half the dose of TGF-ß and SIS3. CONCLUSION: The results indicate that EMT-related biomarkers in serum exosomes can be potentially used for assessing the invasiveness of pituitary adenoma.