Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry - II. Limitations of complex mixture analyses

With an emphasis on obtaining a multitude of high quality tandem mass spect rometry spectra for protein identification, instrumental parameters are des cribed for the liquid chromatography-tandem mass spectrometry analysis of t rypsin digested unfractionated urine using a hybrid quadrupole-time-of-flig ht (Q-TOF) mass spectrometer. Precursor acquisition rates of up to 20 disti nct precursors/minute in a single analysis were obtained through the use of parallel precursor selection (four precursors/survey period) and variable collision induced dissociation integration time (1 to 6 periods summed). Ma ximal exploitation of the gas phase fractionated ions was obtained through the use of narrow survey scans and iterative data-dependent analyses incorp orating dynamic exclusion. The impact on data fidelity as a product of data -dependent selection of precursor ions from a dynamically excluded field is discussed with regards to sample complexity, precursor selection rates, su rvey scan range and facile chemical modifications. Operational and post-ana lysis strategies are presented to restore data confidence and reconcile the greatest number of matched spectra.