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Updated: Dec 10, 2025
Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
A reference measurement of circulating ATPase inhibitory factor 1 (IF1) in humans by LC-MS/MS: Comparison with
Annelise Genoux1, Thibaut Duparc2, Jean-Bernard Ruidavets3
1Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France; University of Toulouse, UMR1048, Paul Sabatier University, Toulouse, France; Service de Biochimie, Pôle de biologie, Hôpital de Purpan, CHU de Toulouse, Toulouse, France.
Insights
ATPase inhibitory factor 1 (IF1) is a potential cardiovascular disease biomarker. A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method accurately quantifies IF1 in human plasma, showing lower levels in coronary heart disease patients.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Biomarker Discovery
Background:
- ATPase inhibitory factor 1 (IF1) is a protein that regulates ATP synthase activity.
- Circulating IF1 levels are associated with HDL-cholesterol and mortality in coronary heart disease (CHD) patients.
- Previous quantification of IF1 used a non-standardized competitive enzyme-linked immunosorbent assay (ELISA).
Purpose of the Study:
- To validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for accurate IF1 quantification in human plasma.
- To compare the performance of the LC-MS/MS method with the existing ELISA method.
- To clinically validate the LC-MS/MS method by assessing its correlation with HDL-cholesterol and its association with CHD.
Main Methods:
- Development and validation of an LC-MS/MS method for IF1 quantification in human plasma after trypsin digestion.
- Validation was performed over 4 independent experiments within the 100-1500 ng/mL range.
- Comparison of LC-MS/MS results with ELISA in 204 individuals from the GENES study.
Main Results:
- The LC-MS/MS method demonstrated high accuracy (95-102%) and low variability (intra-assay <14.2%, inter-assay <14.2%).
- LC-MS/MS and ELISA methods showed strong correlation (r=0.89, p<0.0001) with no significant statistical differences.
- IF1 plasma levels positively correlated with HDL-cholesterol (r=0.38, p<0.0001) and were lower in CHD patients compared to controls (431 ± 132 ng/mL vs 555 ± 173 ng/mL, p<0.0001).
Conclusions:
- The presented LC-MS/MS method is a highly specific and accurate strategy for quantifying IF1 in human plasma.
- This LC-MS/MS method could serve as a reference method for IF1 quantification.
- Validated IF1 quantification supports its role as a promising biomarker for cardiovascular disease.
Abstract:
ATPase inhibitory factor 1 (IF1) is a 9.5 kDa protein that binds to mitochondrial and plasma membrane ATP synthase and selectively inhibits ATP hydrolysis. Recently, IF1 was identified in systemic circulation in humans. IF1 appeared as an independent determinant of HDL-cholesterol with lower levels in coronary heart disease (CHD) patients. Moreover, IF1 was also found to negatively associate with mortality in these patients, supporting the notion that circulating IF1 could be a promising biomarker of cardiovascular disease. However, in previous studies, IF1 was quantified by a non-standardized competitive enzyme-linked immunosorbent assay (ELISA). Herein, we have validated a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) enabling the accurate quantification of IF1 in human plasma. Plasma IF1 was trypsin-digested through an optimized procedure before LC-MS/MS analysis. The method was successfully validated over 4 independent experiments into the range of 100-1500 ng/mL. Intra- and inter-assay variation coefficients had never exceeded 14.2% and accuracy ranged between 95% and 102% for the selected EAGGAFGK peptide marker. Subsequently, the results of the LC-MS/MS method were compared with those obtained using ELISA in 204 individuals from the GENES study. We found that IF1 plasma levels obtained using both techniques were strongly correlated (r = 0.89, p < 0.0001), while the Bland-Altman plot did not indicate any major statistically significant differences. To clinically validate LC-MS/MS, we confirmed the positive correlation between IF1 plasma levels and HDL-cholesterol (r = 0.38, p < 0.0001). Besides, we found lower IF1 plasma levels in CHD patients compared to controls (431 ± 132 ng/mL and 555 ± 173 ng/mL, respectively; p < 0.0001). Hence, it can be concluded that the presented LC-MS/MS analytical method provides a highly specific strategy for IF1 quantification in human plasma and could be proposed as a reference method.
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