Preparation and Analysis of Proteins and Peptides Using MALDI TOF/TOF Mass Spectrometry

Keyur A. Dave1, Madeleine J. Headlam1, Tristan P. Wallis2, Jeffrey J. Gorman1

1 PO Royal Brisbane Hospital, Herston, Queensland, Australia, 2 Mater Adult's Hospital, South Brisbane, Queensland, Australia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.13
DOI:  10.1002/0471140864.ps1613s63
Online Posting Date:  February, 2011
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Abstract

Matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry (MALDI‐TOF/TOF‐MS) is a valuable tool for the analysis of peptides and proteins. Particularly useful features include high sensitivity, fast data acquisition, ease of use, and robust instrumentation. Although MALDI is relatively tolerant to buffers and other impurities, substantial sensitivity enhancement can be achieved through removal of non‐analyte components of samples. Therefore, sample processing to remove buffers and impurities can greatly improve the quality of results obtained by MALDI experiments. This unit describes optimized procedures for enzymatic digestion, preparation of MALDI target plates, thin layer matrix preparation, on‐target sample cleanup, and capillary HPLC‐MALDI co‐spotting of analyte and matrix. Procedures are also described for analysis of on‐membrane proteins by MALDI‐TOF/TOF‐MS before tryptic digestion. Some of these procedures are also applicable to protein spots from two‐dimensional (2‐D) gels. Guidance is also provided for acquisition and interpretation of MS and MS/MS spectra. Curr. Protoc. Protein Sci. 63:16.13.1‐16.13.21. © 2011 by John Wiley & Sons, Inc.

Keywords: protein isolation; purification; digestion and on‐plate cleanup for sensitive MALDI‐TOF/TOF analysis of peptides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Efficient Proteolysis of Proteins by Methanol Co‐Precipitation of Trypsin
  • Basic Protocol 2: Thin‐Layer Preparation of α‐Cyano‐Hydroxy‐Cinnamic Acid (α‐CHCA) on Stainless‐Steel Anchorchip Maldi Target Plates
  • Basic Protocol 3: Desalting and Concentration of Samples by On‐Plate Wash on a Stainless‐Steel Anchorchip Maldi Target Plate Using a Thin‐Layer Matrix Preparation And MALDI‐TOF/TOF Acquisition
  • Alternate Protocol 1: Matrix Deposition After Sample Spotting and Subsequent On‐Plate Washing To Desalt and Concentrate Protein Digests
  • Basic Protocol 4: Co‐Application of Matrix and Cap‐HPLC Eluate onto MALDI target Plate for LC‐Maldi‐TOF/TOF Analysis of Protein Digests
  • Basic Protocol 5: MALDI‐MS Analysis of On‐Membrane Protein on a Polished Steel MALDI Target Plate
  • Basic Protocol 6: Interpretation of MALDI‐TOF/TOF Spectra
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Efficient Proteolysis of Proteins by Methanol Co‐Precipitation of Trypsin

  Materials
  • Concentrated protein sample
  • 1 µg/µl trypsin stock (see recipe)
  • Methanol (ChromAR‐grade, Mallinckrodt Baker), –20°C
  • Nitrogen source
  • 1 M triethylammonium bicarbonate (TEAB; Sigma‐Aldrich)
  • 1.5‐ml safe‐lock microcentrifuge tubes
  • Vortexer
  • Refrigerated microcentrifuge (Eppendorf)
  • Fume hood
  • 37°C water bath

Basic Protocol 2: Thin‐Layer Preparation of α‐Cyano‐Hydroxy‐Cinnamic Acid (α‐CHCA) on Stainless‐Steel Anchorchip Maldi Target Plates

  Materials
  • 100% methanol
  • Milli‐Q water
  • Saturated α‐CHCA solution (see recipe)
  • MTP anchorchip 600/384 (part no. 209513) MALDI target plates (600‐µm spot size/384 sample anchors) (Bruker Daltonics)
  • Lint‐free wipes
  • Sonicator

Basic Protocol 3: Desalting and Concentration of Samples by On‐Plate Wash on a Stainless‐Steel Anchorchip Maldi Target Plate Using a Thin‐Layer Matrix Preparation And MALDI‐TOF/TOF Acquisition

  Materials
  • Sample
  • 99.9% (v/v) trifluoroacetic acid (TFA)
  • Ammonium dihydrogen phosphate (APT buffer; see recipe), ice cold
  • Peptide calibration standards (Bruker Daltonics)
  • Nitrogen source
  • Recrystallization buffer (see recipe)
  • MTP Anchorchip 600/384 (part no. 209513) MALDI target plates (600‐µm spot size/384‐sample anchors) (Bruker Daltonics)
  • UltraFlex III MALDI‐TOF/TOF instrument (Bruker Daltonics)
  • Bruker Daltonics Flex‐series software (FlexControl and FlexAnalysis version 2.4) and Biotools version 3.1

Alternate Protocol 1: Matrix Deposition After Sample Spotting and Subsequent On‐Plate Washing To Desalt and Concentrate Protein Digests

  • Matrix buffer (see recipe)

Basic Protocol 4: Co‐Application of Matrix and Cap‐HPLC Eluate onto MALDI target Plate for LC‐Maldi‐TOF/TOF Analysis of Protein Digests

  Materials
  • 100% (v/v) isopropyl alcohol
  • Nitrogen source
  • 99.9% (v/v) trifluoroacetic acid (TFA)
  • Matrix buffer (see recipe)
  • Peptide calibration solution (Bruker Daltonics)
  • High‐purity organic solvents (ChromAR grade, Mallinckrodt Baker)
  • Massive target plate (MTP) 384 polished steel MALDI target plates (Bruker Daltonics, cat. no. 209520)
  • MALDI plate cleaning kit for stainless steel or gold plates (Applied Biosystems, cat. no. 4342532)
  • Lint‐free tissues
  • Ultimate 3000 capillary HPLC system (Dionex)
  • Acclaim pepmap C18 trap (300‐µm i.d. × 5 mm, 5‐µm particle size, 10‐Å pores; Dionex, cat. no. 160454)
  • Vydac Everest capillary column (150‐µm i.d. × 150‐mm, 5‐µm particle size, 300‐Å pores; Grace, cat. no. 238EV5.1515)
  • Probot micro‐fraction collector (Dionex LC‐Packings)
  • MicroTee for MicroTight sleeves (Upchurch Scientific, cat. no. P‐775)
  • UltraFlex III MALDI‐TOF/TOF MS instrument (Bruker Daltonics)
  • Bruker Daltonics Flex‐series software (FlexControl and FlexAnalysis version 2.4), Biotools version 3.1 and WARP‐LC 1.1

Basic Protocol 5: MALDI‐MS Analysis of On‐Membrane Protein on a Polished Steel MALDI Target Plate

  Materials
  • Destained nitrocellulose membrane with bound protein
  • 3:1 (v/v) acetonitrile/methanol
  • 99.9 % (v/v) trifluoroacetic acid (TFA)
  • DHAP matrix (see recipe)
  • Protein calibration standard I (Bruker Daltonics)
  • 0.6‐ml safe‐lock microcentrifuge tubes (Eppendorf)
  • UltraFlex III MALDI‐TOF/TOF instrument (Bruker Daltonics)
  • Bruker Daltonics Flex‐series software (FlexControl and FlexAnalysis version 2.4)
  • Additional reagents and equipment for preparing MALDI target (see protocol 5)

Basic Protocol 6: Interpretation of MALDI‐TOF/TOF Spectra

  Materials
  • UltraFlex III MALDI‐TOF/TOF instrument (Bruker Daltonics)
  • Bruker Daltonics Flex‐series software (FlexControl and FlexAnalysis version 2.4) and Biotools version 3.1
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Figures

Videos

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Key References
   Walker, J.M. 2002. The Protein Protocols Handbook, Second Edition. Humana Press, Totowa, NJ.
  Details protein isolation and purification procedures from various contributors.
   Aebersold et al., 2003. See above.
  A comprehensive review of mass spectrometry technologies and their application in protein analysis.
   Roepstorff, P., 1996. See above.
  Describes the history of development of MALDI mass spectrometry.
   Schuerenberg et al., 2000. See above.
  An extensive overview of the development of pre‐structured MALDI sample support and its applications.
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