Protein Identification

Protein Identification by MALDI-TOF (PMF)
Proteins are digested with trypsin (cleaves at R-X and K-X except when X is Pro) and a peptide mass fingerprint produced by analysing the digested protein with a MALDI-TOF MS. The monoisotopic masses of the peptides seen in the MALDI-TOF mass spectrum are software selected and used to search a protein, or translated DNA/EST database. The database has been theoretically digested with trypsin and the experimentally generated mass list is compared to theoretically digested database. The match is scored on number of factors, depending on the search program utilised.

Recommended for:

  • The identification of known purified proteins.

Advantages:

  • Rapid analysis and turn around time
  • High sensitivity
  • Cheap
  • Suitable for large numbers of samples

Disadvantages:

  • Protein must be in the database
  • Generally not suitable for proteins <15kDa in size
  • Match based on peptide masses, not sequence information
  • Generally only able to suggest post-translational modifications

See the following pages for Programs used to search MALDI-TOF Peptide Mass Fingerprint data:

Protein Identification by MALDI-TOF/TOF (PMF + MS/MS)

 Proteins are digested in the same manner as for peptide mass fingerprinting and the sample is then analysed by MALDI-TOF, generating a peptide mass fingerprint for the protein. The most abundant peptide ions are then subjected to MALDI-TOF/TOF analysis, providing information that can be used to determine the sequence. The results from both types of analysis are combined and searched using software (e.g. Mascot) against protein, DNA or EST databases, to identify the protein.

Recommended for:

  • The identification of known purified proteins and the analysis of samples requiring a higher level of confidence in identification than can be obtained by MALDI-TOF.

Advantages:

  • Rapid analysis and turn around time (similar to MALDI-TOF) 
  • High sensitivity 
  • Relatively inexpensive 
  • Suitable for large numbers of samples 
  • Able to identify 2-3 proteins in the same spot 
  • Sequence information provides confirmation of peptide mass fingerprint identification 
  • Sequence information allows identification of small proteins (<15kDa)

Disadvantages:

  • Sequence information generally not as complete as that provided by LC/MS/MS 
  • Limited success in identification of proteins that are not in the database

For further details or advice on this APAF service please contact us at ms@proteome.org.au.

To complete a smaple request form for MALDI analysis click here

 

Contact Us

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  • APAF Ltd
  • Level 1, 3 Innovation Road
  • Macquarie University NSW 2109
  • Australia
  • Tel: +61 2 9805 3175
  • Fax: +61 2 9805 3101
  • Email: lwoods@proteome.org.au

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