Predigested Bovine Serum Albumin (Bruker, Billerica, MA) was used as a representative for a simple proteomics sample (25 fmol/µl).
HeLa cell lysate was prepared according to Beck et al.¹ and acidified with formic acid to a final concentration of 200ng/µl. For the simple sample, the tryptic peptides were separated on a novel nano-UHPLC system using a 10min reversed phase gradient (2% - 35% solvent B in 10min. - 0.1% FA in ACN), on standardized 15cm 75µm bore column with C18 resin. The HeLa sample was separated with a reversed phase gradient (from 2% -24% in 90min, 24-48 in 10min. solvent B - 0.1% FA in ACN), on a standardized 40cm 75µm bore columns with C18 resin. Data was acquired with an impact II QTOF MS (Bruker Daltonik, Bremen, Germany) equipped with a CaptiveSpray ion source.
 Beck, S., Michalski, A., Raether, O, Lubeck, M., Kaspar, S., Goedecke, N., Baessmann, C., Hornburg, D, Meier, F., Paron, I., Kulak, N.A., Cox, J. and Mann, M. “The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.” Mol Cell Proteomics. 2015 Jul;14(7):2014-29.