FGF-21 biomarker detection at the sub-nanogram per mL level in human serum using normal-flow liquid chromatography/tandem mass spectrometry
- 30 July 2020
- journal article
- research article
- Published by Wiley in Rapid Communications in Mass Spectrometry
- Vol. 34 (14), e8817
- https://doi.org/10.1002/rcm.8817
Abstract
Rationale Quantitative detection of the FGF-21 biomarker at the sub-nanogram per mL level in human serum has generally been achieved using nanoflow liquid chromatography/tandem mass spectrometry (LC/MS/MS) due to its high sensitivity. However, a nano-LC/MS/MS-based assay can suffer from limited reproducibility and MS signal instability making it challenging to employ it as a robust analytical method for routine clinical applications. Methods To tackle these limitations, parallel reaction monitoring (PRM)-based targeted protein quantification using normal-flow liquid chromatography coupled with high-resolution, accurate mass instrumentation was evaluated as a possible alternative. Different from the conventional selected reaction monitoring (SRM) using triple quadrupole MS, the proposed strategy used high-resolution orbitrap MS coupled with conventional normal-flow liquid chromatography. The primary goal of this assay development effort is to significantly improve the robustness of the LC/MS/MS-based assay while maintaining high sensitivity by the use of high-resolution MS and a large sample loading volume. Results The performance of the normal-flow LC/MS/MS assay was evaluated by using it to quantify the FGF-21 protein, a potential biomarker for non-alcoholic fatty liver disease, in serum samples. Multiple replicated PRM sample quantification results demonstrated the excellent reproducibility and operational robustness of the assay. A limit of quantification of less than 0.4 ng/mL for FGF-21 in a complex serum matrix could be achieved by using the heavy-isotope-labeled peptide technique, a result which is comparable with the sensitivity obtained using the nano-LC/SRM MS-based assay. Conclusions The strategy offered an effective alternative to nano-LC/SRM MS for the quantification of protein biomarkers in a complex biomatrix with much improved reproducibility and operational robustness.Funding Information
- Ningbo University
- National Basic Research Program of China (2017YFC1001700)
- National Natural Science Foundation of China (61971248)
- Ningbo Municipal Bureau of Science and Technology (2018B10075)
This publication has 29 references indexed in Scilit:
- A Targeted Mass Spectrometry Strategy for Developing Proteomic Biomarkers: A Case Study of Epithelial Ovarian CancerMolecular & Cellular Proteomics, 2019
- Using data‐independent, high‐resolution mass spectrometry in protein biomarker research: Perspectives and clinical applicationsProteomics – Clinical Applications, 2014
- Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serumProceedings of the National Academy of Sciences of the United States of America, 2012
- On the Development of Plasma Protein BiomarkersJournal of Proteome Research, 2011
- Perspectives of targeted mass spectrometry for protein biomarker verificationCurrent Opinion in Chemical Biology, 2009
- The interface between biomarker discovery and clinical validation: The tar pit of the protein biomarker pipelineProteomics – Clinical Applications, 2008
- High Sensitivity Detection of Plasma Proteins by Multiple Reaction Monitoring of N-GlycositesMolecular & Cellular Proteomics, 2007
- Protein biomarker discovery and validation: the long and uncertain path to clinical utilityNature Biotechnology, 2006
- Biomarkers in Cancer Staging, Prognosis and Treatment SelectionNature Reviews Cancer, 2005
- The Human Plasma ProteomeMolecular & Cellular Proteomics, 2002