Six bovine protein digest

Splitless, nano-flow liquid chromatography separation was performed using a Waters nanoACQUITY (Waters Corp., Milford, MA) UPLC system configured for partial loop injection coupled online to the Q-Exactive mass spectrometer. Fused silica capillary tubing (75 μm I.D.; Polymicro Technologies) was pulled to a tip of ~ 5 μm at one end and packed with 15 cm of Jupiter Proteo 4 μm C12 beads (Phenomenex, Torrance, CA). A Kasil fritted trap was prepared using a 100-μm I.D. capillary tube packed with 2 cm of the identical C12 packing material, and 2 μL of sample was trapped at a flow rate of 1 μL/min for 8 min prior to going in-line with the packed tip. Peptides were separated over a 60-min linear reversed phase gradient ranging from 2 to 32% Buffer B (0.1% w/v formic acid in acetonitrile) mixed with Buffer A (0.1% w/v formic acid in water) with a flow rate of 300 nL/min. Quality control runs analyzed a six bovine protein digest (Bruker-Michrom, Auburn, CA) using a 30-min linear gradient.

We generated a series of samples in which an equimolar six bovine protein digest (Bruker-Michrom) was spiked into a background matrix of digested S. cerevisiae soluble lysate. The yeast tryptic digest background was prepared from 400 μL of 5.1 μg/μL of yeast proteins (strain BY4741) that are soluble in 50 mM ammonium bicarbonate (ABC). This digest was combined with 60 μL 1 M ABC, 110 μL 0.5% PPS detergent (Agilent Technologies, Santa Clara, CA), and 6 μL 500 mM Tris(2-carboxyethyl)phosphine (Thermo Fisher Scientific, Rockford, IL) for 30 min at 60 °C. The reaction was cooled to room temperature and 8 μL 500 mM iodoacetamide was allowed to react for 30 min prior to the addition of 40 μL of 2 μg/μL TPCK-treated trypsin (Worthington Biochemical, Lakewood, NJ). Digestion proceeded for 4 h at 37 °C. Residual iodoacetamide was quenched with 5 μL 500 mM dithiothreitol for 15 min at 37 °C. The final digest was acidified with 60 μL 10% trifluoroacetic acid. The six bovine protein tryptic peptide mixture (Bruker, Auburn, CA) was mixed in various concentrations (48, 16, 4, 2, 1, 0.5, 0.25, 0.05, and 0.025 fmol/μL) with a constant yeast tryptic peptide background (0.75 μg/μL). Two microliters of each sample was injected for each mass spectrometry run.