A reference measurement of circulating ATPase inhibitory factor 1 (IF1) in humans by LC-MS/MS: comparison with conventional ELISA

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 100-1,500 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 method provides a highly specific strategy for IF1 quantification in human plasma and could be proposed as a reference technique. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/20097816v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@19e95f7org.highwire.dtl.DTLVardef@87bf40org.highwire.dtl.DTLVardef@1d0a33org.highwire.dtl.DTLVardef@88d4cc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIATPase inhibitory factor 1 (IF1) is a biomarker of coronary heart disease in humans. C_LIO_LIThus far, IF1 plasma concentrations have been determined by non-standardized competitive ELISA. C_LIO_LIHere, we have developed and validated an LC-MS/MS assay for the accurate IF1 quantification in human plasma. C_LIO_LIOur LC-MS/MS assay is rapid and incurs reasonable costs, thus making it amenable for clinical practice. C_LI