So far, mass spectrometry based targeted proteomics is the most sensitive approach to answer and address specific biological questions in an accurate and quantitative fashion. However, the data analysis design used for such quantification varies in the field leading to discrepancies in the reported values. In this study, different quantification strategies based on calibration curves were evaluated and compared. The best accuracy and coefficient of variation was achieved by ratio to ratio calibration curves. We applied the ratio to ratio quantification approach to analyze very low abundant insulin signaling proteins such as PIK3RA (0.10-0.93 fmol/μg), AKT1 (0.1-0.39 fmol/μg) and the Insulin receptor (0.22 -2.62 fmol/μg) in a fat cell model and demonstrated the adaptation of this pathway at different states of insulin sensitivity.

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