Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry

Andrew J. Link1, Michael P. Washburn2

1 Vanderbilt University School of Medicine Nashville, Tennessee, 2 Stowers Institute for Medical Research, Kansas City, Missouri
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.1
DOI:  10.1002/0471140864.ps2301s78
Online Posting Date:  November, 2014
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Abstract

Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in‐gel digestions by directly identifying protein mixtures in solution. One method used extensively is named Multidimensional Protein Identification Technology (MudPIT), where reversed‐phase chromatography and strong cation–exchange chromatography are coupled directly in a microcapillary column. This column is then placed in line between an HPLC and a mass spectrometer for complex mixture analysis. MudPIT remains a powerful approach for analyzing complex mixtures like whole proteomes and protein complexes. MudPIT is used for quantitative proteomic analysis of complex mixtures to generate novel biological insights. © 2014 by John Wiley & Sons, Inc.

Keywords: proteomics; multidimensional protein identification technology; direct analysis of large protein complexes; protein complexes; chromatography

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Off‐Line SCX Fractionation Coupled to RP‐LC‐ESI‐MS/MS
  • Basic Protocol 2: Multidimensional Protein Identification Technology (MudPIT)
  • Support Protocol 1: Constructing Fritted Capillary HPLC Columns
  • Support Protocol 2: Constructing Fritless Capillary HPLC Columns
  • Support Protocol 3: Packing Capillary HPLC Columns
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Off‐Line SCX Fractionation Coupled to RP‐LC‐ESI‐MS/MS

  Materials
  • 2% and 95% acetonitrile/0.1% TFA
  • 70% formic acid/30% isopropanol
  • Enzymatically digested or chemically cleaved protein sample
  • 0.5% acetic acid
  • SCX buffers A, B, C, and D (see reciperecipes)
  • Angiotensin solution (see recipe)
  • High‐pressure and ultrapure nitrogen
  • Trapping system:
    • 0.5‐, 20‐, or 200‐μg reversed‐phase (RP) trapping cartridges (Microm BioResources)
    • RP trapping cartridge holder with syringe port and capillary fitting (Microm BioResources)
  • 10‐ and 50‐μl syringes (Hamilton)
  • SCX apparatus:
    • 50‐ and 75‐μm‐i.d. × 365‐μm‐o.d. fused‐silica capillary (FSC; Polymicro Technology)
    • Quaternary HPLC pump (Agilent Technologies)
    • PEEK micro tee (Upchurch)
    • PEEK microtight adapter (Upchurch)
    • PEEK 380‐μm‐i.d. microtight sleeves (Upchurch)
    • Capillary SCX column (see protocol 5)
    • PEEK microtight true zero dead volume (ZDV) union (Upchurch)
    • Syringe port (Upchurch)
    • 10‐μl sample loop (Upchurch)
  • Quartz capillary cleaving tool (Scientific Instrument Services)
  • Reversed‐phase (RP) apparatus:
    • Binary HPLC pump (Agilent Technologies)
    • PEEK micro tee (Upchurch)
    • PEEK 380‐μm‐i.d. microtight sleeves (Upchurch)
    • Capillary RP column (see protocol 5)
    • 50‐ and 75‐μm‐i.d. × 365‐μm‐o.d. fused‐silica capillary (FSC; Polymicro Technology)
    • 0.025‐in.‐o.d. gold wire (Scientific Instrument Services)
  • Packing vessel (CorSolutions)
  • 1/16‐in. to 0.4‐mm Vespel ferrules (Chromatography Research Supplies)
  • 20‐μm‐i.d. × 365‐μm‐o.d. uncoated emitter tip with 10‐μm opening (New Objective)
  • 5‐μl graduated glass capillary (Sigma)
  • Microspray ionization source with xyz manipulator
  • ESI mass spectrometer
  • Additional equipment and reagents for analyzing MS/MS data (unit )

Basic Protocol 2: Multidimensional Protein Identification Technology (MudPIT)

  Materials
  • MudPIT buffers A, B, C, and D (see reciperecipes)
  • Enzymatically digested or chemically cleaved protein sample
  • Quaternary HPLC pump (Agilent Technologies) and Xcalibur software (Thermo Fisher Scientific)
  • PEEK micro‐cross (Upchurch)
  • PEEK 380‐μm‐i.d. microtight sleeves (Upchurch)
  • Triphasic MudPIT column (see protocol 4)
  • 50‐μm‐i.d. × 365‐μm‐o.d. fused‐silica capillary (Polymicro Technology)
  • 0.025‐in.‐o.d. gold wire (Scientific Instrument Services)
  • ESI voltage source
  • 5‐μl graduated glass capillary pipet
  • Quartz capillary cleaving tool (Scientific Instrument Services)
  • Packing vessel (CorSolutions)
  • 1/16‐in. to 0.4‐mm Vespel ferrules (Chromatography Research Supplies)
  • LTQ or Orbitrap mass spectrometer (Thermo Fisher Scientific)
  • Microspray ionization source with xyz manipulator
  • Additional equipment and reagents for loading from a pneumatic vessel (see protocol 1, steps 38 to 42) and analyzing MS/MS data (unit )

Support Protocol 1: Constructing Fritted Capillary HPLC Columns

  Materials
  • Methanol
  • High‐pressure nitrogen
  • Potassium silicate (KASIL #1; The PQ Corporation)
  • Formamide
  • 1 M ammonium nitrate
  • 1 M HCl
  • Acetonitrile
  • 75‐μm‐i.d. × 365‐μm‐o.d. fused‐silica capillary (FSC; Polymicro Technology)
  • Quartz capillary tubing cleaving tool (Chromatography Research Supplies)
  • Packing vessel (CorSolutions)
  • 1/16‐in. to 0.4‐mm Vespel ferrules (Chromatography Research Supplies)
  • Stereomicroscope
  • 100°C heat block

Support Protocol 2: Constructing Fritless Capillary HPLC Columns

  Materials
  • Methanol
  • 100‐μm‐i.d. × 365‐μm‐o.d. fused‐silica capillary (FSC; Polymicro Technology)
  • Alcohol lamp
  • P‐2000 laser micropipet puller (Sutter Instruments)
  • Quartz capillary tubing cleaving tool (Chromatography Research Supplies)

Support Protocol 3: Packing Capillary HPLC Columns

  Materials
  • Methanol
  • Reverse‐phase (RP) packing material (Phenomenex Aqua 5 μm C18 125A)
  • Strong cation–exchange (SCX) packing material (Phenomenex Luna 5μm SCX 100A)
  • High‐pressure and ultrapure nitrogen gas
  • MudPIT Buffer A (see recipe)
  • 1.8‐ml glass vial (Chromatography Research Supplies)
  • Sonicator
  • Packing vessel (CorSolutions)
  • 1/16‐in. to 0.4‐mm Vespel ferrules (Chromatography Research Supplies)
  • Empty fritted or fritless column (see Support Protocol protocol 31 or protocol 42)
  • Stereomicroscope
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Figures

Videos

Literature Cited

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   Farley, A.R. , Powell, D.W. , Weaver, C.M. , Jennings, J.L. , and Link, A.J. 2011. Assessing the components of the eIF3 complex and their phosphorylation status. J. Proteome Res. 10:1481‐1494.
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Key References
   Fournier et al., 2007. See above.
  This is a detailed and extensive review on multidimensional separations and proteomics.
   Kinter, M. and Sherman, N.E. 2000. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley‐Interscience, New York.
  This is an excellent, easy‐to‐read tutorial on mass spectrometry for identifying proteins and post‐translational modifications. Many of the fundamental applications in this unit are described in detail from the view of the bench scientist.
   Link et al., 1999. See above.
  This publication demonstrates the original biphasic MudPIT approach and the use of multidimensional technology to directly analyze protein complexes.
   Wolters et al., 2001. See above.
  This technical publication optimizes and automates MudPIT for analysis of complex peptide mixtures.
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