MacCoss - Improving Precursor Selectivity in Data Independent Acquisition Using Overlapping Windows

Welcome to the Panorama repository for the manuscript:

Improving Precursor Selectivity in Data Independent Acquisition Using Overlapping Windows

Dario Amodei*1, Jarrett Egertson*2, Brendan MacLean2, Richard Johnson2, Gennifer E Merrihew2, Austin Keller2, Don Marsh2, Olga Vitek3, Parag Mallick&1, Michael J. MacCoss&2

1. Department of Radiology, Stanford University, 3155 Porter Drive, Palo Alto, CA 

2. Department of Genome Sciences, University of Washington, 3720 15th Ave. NE, Seattle, WA

3. College of Computer and Information Science, Northeastern University, 440 Huntington Ave, Boston, MA

Please find the manuscript abstract and experiment description below.

To access raw data and Skyline files for the spike-in and rapamycin-treatment experiments detailed in the manuscript, click the applicable folder in the Projects section below.

A tutorial on generating an overlapping window isolation list using Skyline can be found here.

A tutorial on demultiplexing overlap data using MSConvert can be found here.

Improving Precursor Selectivity in Data Independent Acquisition Using Overlapping Windows

  • Organism: Saccharomyces cerevisiae, Bos taurus
  • Instrument: Q Exactive
  • SpikeIn: Yes
  • Keywords: yeast, rapamycin, proteasome, multiplexing, data independent acquisition, Q-Exactive, orbitrap, spike-in, quantification, bovine
  • Lab head: Michael MacCoss
A major goal of proteomics research is the accurate and sensitive identification and quantification of a broad range of proteins within a sample. Data Independent Acquisition (DIA) approaches that acquire MS/MS spectra independently of precursor information have been developed to overcome the reproducibility challenges of data-dependent acquisition and the limited breadth of targeted proteomics strategies. Typical DIA implementations use wide MS/MS isolation windows to acquire comprehensive fragment ion data. However, wide isolation windows produce highly chimeric spectra, limiting the achievable sensitivity and accuracy of quantification and identification. Here we present a DIA strategy in which spectra are collected with overlapping, (rather than adjacent or random) windows and then computationally demultiplexed. This approach improves precursor selectivity by nearly a factor of 2, without incurring any loss in mass range, mass resolution, chromatographic resolution, scan speed, or other key acquisition parameters. We demonstrate a 64% improvement in sensitivity and a 17% improvement in peptides detected in a 6 protein bovine mix spiked into a yeast background. To confirm the method’s applicability to a realistic biological experiment, we also analyze the regulation of the proteasome in yeast grown in rapamycin and show that DIA experiments with overlapping windows can help elucidate its adaptation toward the degradation of oxidatively damaged proteins. Our integrated computational and experimental DIA strategy is compatible with any DIA-capable instrument. The computational demultiplexing algorithm required to analyze the data has been made available as part of the open-source proteomics software tools Skyline and msconvert (Proteowizard), making it easy to apply as part of standard proteomics workflows.
Experiment Description
This project consists of two experiments -- a bovine -spike in experiment, and a rapamycin-treatment experiment. They are described separately here. BOVINE EXPERIMENT: 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. |||||||||||| RAPAMYCIN EXPERIMENT Three conditions (WT, WT + rapamycin, tor1) were run with three biological replicates each. For each of these runs, three data acquisitions methods were run (20 m/z overlap, 10 m/z, and DDA).
Sample Description
This project consists of two experiments -- a bovine -spike in experiment, and a rapamycin-treatment experiment. Samples for each experiment are described separately here: BOVINE EXPERIMENT 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. ||||||||||| RAPAMYCIN EXPERIMENT Three conditions were tested in this experiment: wild type yeast, wild type yeast grown in rapamycin, and tor1 deletion mutant yeast. The wild type and tor1 haploid strains of S. cerevisiae were from an ORF deletion collection33 and had the parental background BY4742 -- MATα hist3∆1 leu2∆0 lys2∆0 ura3∆0. Both strains were grown in YPD media in three biological replicates. The wild type strain was grown either in the presence or absence of 10 nM rapamycin. Strains were grown from OD600 ~0.15 to OD600 0.6 prior to lysis and digestion. The samples without rapamycin grew to this density in about 6 hours, while the rapamycin treated strains required 8 hours. Cells were lysed by bead beating using a lysis buffer of 50 mM ammonium bicarbonate at pH 7.8 with phosphatase inhibitors (Pierce – Halt phosphatase inhibitor cocktail). Bead beating was for one minute, followed by one minute of cooling the sample on ice, repeated three times. The digestion was performed for one hour using sequencing-grade trypsin (Promega, Madison, WI) as in Hoopmann et. al.34 except using 0.1% PPS Silent Surfactant (Protein Discoveries) instead of RapiGest SF (Waters Corporation, Milford, Ma).
Created on 11/30/18 10:27 AM