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

• Main Authors: Christina Looße, Sara Galozzi, Linde Debor, Mattijs K. Julsing, Bruno Bühler, Andreas Schmid, Katalin Barkovits, Thorsten Müller, Katrin Marcus
• Format: Article
• Language: English
• Published: Elsevier 2015-06-01
• Series: EuPA Open Proteomics
• Subjects: Direct infusion; Quantification; Single ion monitoring (SIM); Q Exactive; Cytochrome P450 (CYP)
• Online Access: http://www.sciencedirect.com/science/article/pii/S2212968515000070

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.