Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

Abstract

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.

Keywords

• Direct infusion
• Quantification
• Single ion monitoring (SIM)
• Q Exactive
• Cytochrome P450 (CYP)