UNIT 16.13 Preparation and Analysis of Proteins and Peptides Using MALDI TOF/TOF Mass Spectrometry

  1. Keyur A. Dave1,
  2. Madeleine J. Headlam1,
  3. Tristan P. Wallis2,
  4. Jeffrey J. Gorman1

Published Online: 1 FEB 2011

DOI: 10.1002/0471140864.ps1613s63

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Dave, K. A., Headlam, M. J., Wallis, T. P. and Gorman, J. J. 2011. Preparation and Analysis of Proteins and Peptides Using MALDI TOF/TOF Mass Spectrometry. Current Protocols in Protein Science. 63:16.13:16.13.1–16.13.21.

Author Information

  1. 1

    PO Royal Brisbane Hospital, Herston, Queensland, Australia

  2. 2

    Mater Adult's Hospital, South Brisbane, Queensland, Australia

Publication History

  1. Published Online: 1 FEB 2011
  2. Published Print: FEB 2011


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.


  • protein isolation;
  • purification;
  • digestion and on-plate cleanup for sensitive MALDI-TOF/TOF analysis of peptides