C. Looße, S. Galozzi, L. Debor, M.K. Julsing, B. Bühler, A. Schmid, K. Barkovits, T. Müller, and K. Marcus (2015).
Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples.
EuPA Open Proteomics 7: 20–26.
doi: 10.1016/j.euprot.2015.03.001
Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.