BovineSpikeIn

Experiment Description

Analysis of a bovine protein digest spiked into a yeast matrix background at varying concentrations. Data were analyzed using three methods for DIA acquisition: 10 m/z, 20 m/z, and 20 m/z with overlap. The 10 m/z method was a repeating cycle of twenty 10 m/z wide windows, covering the range from 500 to 700 m/z, as shown in Figure 1A. The 20 m/z method was a repeating cycle of twenty 20 m/z wide windows, covering the range from 500 to 900 m/z, as shown in Figure 1B. The 20 m/z overlap method also used twenty 20-m/z wide windows, but alternating cycles were offset by −10 m/z, so that even-numbered cycles covered windows from 500 to 900 m/z, while odd-numbered cycles covered windows from 490 to 890 m/z (Figure 1C). The 20 m/z overlapping data was analyzed with (demux) and without (overlap) demultiplexing.

Sample Description

We generated a series of samples in which an equimolar 6 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 minutes at 60° C. The reaction was cooled to room temperature and 8 µl 500 mM iodoacetamide was allowed to react for 30 minutes prior to the addition of 40 µl of 2 µg/µl TPCK-treated trypsin (Worthington Biochemical, Lakewood, NJ). Digestion proceeded for 4 hours at 37° C. Residual iodoacetamide was quenched with 5 µl 500 mM dithiothreitol for 15 minutes at 37° C. The final digest was acidified with 60 µl 10% trifluoracetic 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). 2 µL of each sample was injected for each mass spectrometry run.

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20mz_manual.sky.zip2018-11-30 09:51:396373739530
20mz_normalize.sky.zip2018-11-30 09:51:391818183730
demux_manual.sky.zip2018-11-30 09:51:396373739530
demux_normalize.sky.zip2018-11-30 09:51:391848486830
overlap_normalize.sky.zip2018-11-30 09:51:391868689130
10mz_manual.sky.zip2018-11-30 09:51:396373739530
overlap_manual.sky.zip2018-11-30 09:51:396373739530
10mz_normalize.sky.zip2018-11-30 09:51:391848486730