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FGF‐21 biomarker detection at the sub‐nanogram per mL level in human serum using normal‐flow liquid chromatography/tandem mass spectrometry
Author(s) -
Gao Wenqing,
Yang Jiaqian,
Liu Rong,
Yan Yinghua,
Xie Chengyi,
Yu Jiancheng,
Tang Keqi
Publication year - 2020
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.8817
Subject(s) - chemistry , chromatography , reproducibility , detection limit , tandem mass spectrometry , liquid chromatography–mass spectrometry , mass spectrometry , selected reaction monitoring , triple quadrupole mass spectrometer
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.