on 05-12-201702:41 AM - edited on 10-15-202111:32 AM by Closed Account
Joshua J. Nicklay, Kerry M. Hassell, and Aran Paulus ASMS 2017 Poster Purpose: To demonstrate the benefits of running Data Independent Analysis (DIA) by capillary flow LC using a 150 μm ID EASY-Spray column on a Thermo Q Exactive™ HF MS relative to nanoflow LC for proteomics.
Methods: A complex, three proteome mixture of yeast, E. coli and human cell digests was spiked with the Promega 6x5 peptide mixture and the Biognosys iRT peptide calibrants. Peptide spectral libraries were generated by Data Dependent Analysis (DDA) using nanoflow LC. DIA was run using various gradient lengths and sample amounts at both nanoflow rates and capillary flow rates. Library recovery and sensitivity were compared between the two flow regimes.
Results: Running shorter gradients by capillary flow DIA greatly increases the number of proteins and peptides identified per hour relative to nanoflow DIA. Sensitivity as measured by 6 heavy labeled peptides spanning five orders of magnitude is minimally reduced by capillary flow DIA.