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

Publication date: Available online 21 March 2015 Source:EuPA Open Proteomics Author(s): Christina Looße , Sara Galozzi , Linde Debor , Mattijs K. Julsing , Bruno Bühler , Andreas Schmid , Katalin Barkovits , Thorsten Müller , Katrin Marcus 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 E. 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 4.5minutes (that could be further decreased to 30seconds) for a single sample in contrast to 65minutes by the LC-MS methods. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.
Source: EuPA Open Proteomics - Category: Bioinformatics Source Type: research