Application of SWATH Proteomics to Mouse Biology

Yibo Wu1, Evan G. Williams1, Ruedi Aebersold2

1 Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, 2 Faculty of Science, University of Zurich, Zurich
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.28
Online Posting Date:  June, 2017
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The quantitative measurement of the proteome has been shown to yield new insights into physiology and cell biology that cannot be determined from the genome and transcriptome because the quantitative relationship between transcriptome and proteome is complex. MS‐based proteomics techniques, such as SWATH‐MS, have recently advanced to the point at which they may be reliably applied by biologists who are not specialists in mass spectrometry. Here we provide standard protocols for preparation of tissue samples for input into the SWATH‐MS analytical pipeline. These protocols are designed for high‐throughput processing of tissues with ≥5 mg of sample available for analysis. Studies with extremely limited amounts of tissue should consider PCT‐SWATH. An experienced single user should be able to process 48 samples per day for injection into the mass spectrometer, or up to 144 samples a week. The machine time necessary for running these samples with SWATH is approximately 1.5 hr per sample. Data acquisition protocols are also provided. © 2017 by John Wiley & Sons, Inc.

Keywords: proteomics; systems biology; mass spectrometry

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Table of Contents

  • Introduction
  • Basic Protocol 1: Protein Preparation for SWATH‐MS
  • Basic Protocol 2: Peptide Preparation for SWATH‐MS
  • Basic Protocol 3: Data‐Dependent Acquisition and Spectral Library Generation Protocol
  • Basic Protocol 4: Data‐Independent Acquisition and Targeted “Peptide‐Centric” Data Extraction Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Protein Preparation for SWATH‐MS

  • RIPA‐M buffer (see recipe), freshly prepared
  • Urea‐T buffer (see recipe), freshly prepared
  • Tissue sample
  • Protein quantification assay kit: e.g., bicinchoninic acid (BCA) or Bradford
  • Homogenizer (Dounce pestle recommended, but a metallic bead homogenizer can also work)
  • Refrigerated centrifuge (temperature range, ≤4°C; capacity, ≥24 2‐ml tubes; rotation speed, ≥20,000 × g)
  • Bath sonicator (recommended)
  • Vortex mixer

Basic Protocol 2: Peptide Preparation for SWATH‐MS

  • Protein samples (e.g., from protocol 1)
  • Acetone (HPLC‐grade), cooled to −20°C
  • Urea in 0.1 M ammonium bicarbonate (NH 4HCO 3)
  • Ammonium bicarbonate (NH 4HCO 3)
  • Dithioethreitol (DTT)
  • Iodoacetamide (IAA)
  • Trypsin (sequencing‐grade)
  • Methanol (HPLC‐grade)
  • Acetonitrile (ACN; HPLC‐grade)
  • Formic acid (FA; 98% to 100%; see annotation to step 24, below)
  • Trifluoroacetic acid (TFA)
  • Indexed Retention Time peptides (iRT; from Biognosys)
  • Heated shaker plate (37°C)
  • Refrigerated centrifuge (temperature range, ≤4°C; capacity, ≥24 2‐ml tubes; rotation speed, ≥20,000 × g)
  • Silica C18 columns (e.g., MacroSpin Columns from The Nest Group)
  • Vacuum evaporator (e.g., SpeedVac)
  • Vials for mass spectrometry
  • Mass spectrometer
  • Vortex mixer
  • NanoDrop microspectrophotometer
  • Bath sonicator

Basic Protocol 3: Data‐Dependent Acquisition and Spectral Library Generation Protocol

  • Peptide mixture (e.g., from protocol 2)
  • Buffer A: 2% ACN, 0.1% FA solution in H 2O
  • Buffer B: 98% ACN solution with 0.1% FA
  • pH 3‐10 IPG strip (Amersham Biosciences)
  • 3100 OFFGEL Fractionator (Agilent Technologies)
  • Sciex 5600 TripleTOF mass spectrometer
  • Eksigent NanoLC Ultra 2D Plus HPLC (interfaced to the mass spectrometer)
  • C18 column (Magic, 3 μm; from New Objective)
  • Analytical column (e.g, PicoFrit; Thermo Fisher Scientific; with 75‐μm diameter)
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Literature Cited

Literature Cited
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