Label-free protein quantification on tandem mass spectra acquired in a data-independent mode provides accurate measurements over five orders of concentration magnitude in complex matrices

Label free quantification using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is widely used in quantitative proteomics. However, data-dependent bottom-up proteomics suffers from low reproducibility due to semi-random selection of precursor ions for tandem mass spectrometry. In addition, this acquisition mode is biased towards abundant peptides. To overcome these problems, alternative precursor-ion selection methods were developed, such as data-independent acquisition and pseudo-multiple selected reaction monitoring (p-mSRM). With these methods, several tandem mass spectra are acquired over the chromatographic elution time of precursor ions. In this report, we investigated if the acquired tandem mass spectra can be used for label-free quantification. For this, extracted fragment ion currents were correlated to relative protein concentration. A linear relationship between ion current and proteins concentration was observed over five orders of magnitude. Thus, we conclude that relative label-free peptide and protein quantification can be performed in an ion trap using the data-independent acquisition mode.