Mass Spectrometry‐Based Bottom‐Up Proteomics: Sample Preparation, LC‐MS/MS Analysis, and Database Query Strategies

Matthew J. Wither1, Kirk C. Hansen1, Julie A. Reisz1

1 Biological Mass Spectrometry Core, Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.4
DOI:  10.1002/cpps.18
Online Posting Date:  November, 2016
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Recent technological advances in mass spectrometry (MS) have made possible the investigation and quantification of complex mixtures of biomolecules. The exceptional sensitivity and resolving power of today's mass spectrometers allow for the detection of proteins and peptides at low femtomole quantities; however, these attributes demand high sample purity to minimize artifacts and achieve the highest degree of biomolecule identification. Tissue preparation for proteomic studies is particularly challenging due to their heterogeneity in cell type, presence of insoluble biomaterials, and wide diversity of biomolecules. The workflow described herein details sample preparation from tissues through protein extraction, proteolysis, and purification to generate peptides for MS analysis. Increased peptide resolution and a corresponding increase in protein identification is accomplished using polarity‐based fractionation (C18 resin) at the peptide level. Additionally, approaches to instrument set up, including the use of nanoscale liquid chromatography and quadrupole Orbitrap MS, along with database searching, are described. © 2016 by John Wiley & Sons, Inc.

Keywords: LC‐MS/MS; mass spectrometry; peptide sequencing; proteomics; fractionation

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sample Preparation and nanoLC‐MS/MS Analysis
  • Alternate Protocol 1: nanoLC‐MS Parameters for Thermo LTQ Orbitrap Velos Pro AND Eksigent nanoLC Ultra 2D
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Sample Preparation and nanoLC‐MS/MS Analysis

  • Samples/tissues of interest: 3 to 10 mg of dry organ or tissue is generally sufficient; use 2 to 3 times this amount when using wet weight
  • Liquid nitrogen
  • Extraction buffer (see recipe)
  • Protease Inhibitor (e.g., ProteaseArrest; G‐Biosciences, cat. no. 786‐108 or Halt Protease Inhibitor Cocktail, Thermo Scientific, cat. no. 87786]
  • Pierce BCA Protein Assay kit (ThermoFisher Scientific, cat. no. 23225) or Sigma‐Aldrich BCA kit (Sigma‐Aldrich, cat. no. BCA‐1)
  • Urea buffer (see recipe)
  • Dithiothreitol (DTT) solution (see recipe)
  • Iodoacetamide (IAM) solution (see recipe)
  • Ammonium bicarbonate (NH 4HCO 3)
  • Trypsin solution (see recipe)
  • Double‐distilled water
  • Ammonium hydroxide (NH 4OH)
  • Acetonitrile, Optima LC‐MS (Fisher Scientific, cat. no. A955‐4)
  • High‐purity water, such as double‐distilled or commercial (HPLC grade)
  • Acetonitrile with 0.1% formic acid (Optima LC‐MS; Fisher Scientific, cat. no. LS120‐4)
  • Water with 0.1% formic acid (Optima LC‐MS; Fisher Scientific)
  • Ceramic mortar and pestle
  • Analytical Balance (Sartorius, Entris 64‐1 S)
  • 1‐mm glass beads (Next Advance)
  • Bead Beater (Next Advance, Bullet Blender Storm 24)
  • Benchtop centrifuge capable of 14,000 × g (Eppendorf 5424 w/24 place rotor)
  • 10 kDa molecular‐weight cut‐off (MWCO) filters (Sartorius, Vivacon 500, cat. no. VN01H02)
  • Temperature‐controlled incubator
  • Nano‐LC system
  • C18 Spin Tips (Pierce, Thermo Scientific, cat. no. 84850)
  • Autosampler vials (Phenomenex, cat. no. AR0‐9992‐13)
  • Vacuum centrifuge
IMPORTANT NOTE: Sample contamination by ambient material (particularly keratins) is challenging to avoid in proteomic workflows. Keratins are introduced to samples through skin cells, hair, fingerprints, and dust or lint in the laboratory. To avoid introduction of keratin and other contaminants, wear clean gloves and keep samples, pipet tips, and all reagents covered/capped when not in use.
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Literature Cited

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