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Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis.

ABSTRACT: Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay (fvPRM) has the ability to track secreted flaviviral nonstructural protein 1 (NS1) over a broad diagnostic and typing window with high sensitivity, specificity, extendibility, and multiplexing capability. These features, pivotal and pertinent to efficient response toward flavivirus outbreaks, including newly emerging flavivirus strains, circumvent the limitations of current diagnostic assays. fvPRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management.

SUBMITTER: Wee S

PROVIDER: S-EPMC6452653 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis.

Wee Sheena S Alli-Shaik Asfa A Kek Relus R Swa Hannah L F HLF Tien Wei-Ping WP Lim Vanessa W VW Leo Yee-Sin YS Ng Lee-Ching LC Hapuarachchi Hapuarachchige C HC Gunaratne Jayantha J

Proceedings of the National Academy of Sciences of the United States of America 20190318 14

Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay (<i>fv</i>PRM) has the ability to track secreted flavivir ...[more]

PMID: 30886083

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Project description:Background:PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application. Methods:A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories. Results:Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R?=?0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R?=?0.98) demonstrating the robustness of the methodology and ease of method transfer. Conclusions:An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients.

Dataset Information

Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis.

Publications

Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis.

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