Peptide-Mass Fingerprinting

Introduction: Peptide Mass Fingerprinting (PMF) is a technique used to identify proteins by matching their constituent fragment masses (peptide masses) to the theoretical peptide masses generated from a protein or DNA database.  The first step in PMF is that an intact, unknown protein is cleaved with a proteolytic enzyme to generate peptides. With PMF, heterogeneity is most commonly imparted to the unknown protein with a trypsin digestion.  A PMF database search is usually employed following MALDI TOF mass analysis.  The premise of peptide mass fingerprinting is that every unique protein will have a unique set of peptides and hence unique peptide masses.  Identification is accomplished by matching the observed peptide masses to the theoretical masses derived from a sequence database.  PMF identification relies on observing a large number of peptides, 5+, from the same protein at high mass accuracy.  This technique does well with 2D gel spots where the protein purity is high.  PMF protein identification can run into difficulties with complex mixtures of proteins.  Low level ID also becomes difficult due to commonplace contamination by keratin.
                      Figure 1

MALDI TOF Mass Spectrum

Figure 1.  This spectrum was collected on a Voyager DE STR MALDI TOF mass spectrometer and was an average of 240 scans. Peptide peaks appear as [M+H]1+ ions.  The peaks appearing at +22u are sodium adducts.  For a closer look download a PDF of this spectrum, included in the PDF is a close-up of the cluster of peaks around 1850 m/z. 

Discussion William Henzel and co-workers at Genentech Inc., were the first to use this technique to identify proteins in a database(1-2).  But as one can see there were a number of notable researchers working on this technique in the early 90's(2-5). Why so many papers in 1993?  Well, for the most part it was the introduction of a MALDI TOF instrument capable of 50 ppm mass accuracy that made PMF routine.  In MALDI TOF mass spectrometry, peptides appear as singly charged species in the mass spectrum, see Figure 1, this type of spectrum is simple to interpret unlike an electrospray (ESI) mass spectrum which displays multiply charged species.  PMF can be used to identify proteins in ESI spectra but it is seldom used because the peptide masses would need to be deconvoluted for each search(6).  PMF identification relies on high mass accuracy (7-8) and to a greater extent enzyme specificity.  Without a known enzyme specificity PMF identification fails.  For an article on the origins of mass mapping the reader is referred to reference (9)

A Short Exercise: Try performing your own PMF search.  Use the peptide masses from the spectrum in Figure 1 to search a protein database.  Copy the masses from the spectrum above into the Aldente search engine located on the ExPASy Proteomics Server or use the text file we have created for you.  From the text file try pasting the masses into the "peak list area" of  Aldente.  Before starting the search familiarize yourself with the settings. Only a few changes will be needed, make sure the database is set to Swiss-Prot, and taxon is set to "All".  If you would like to see a screen shot of the data entry page when we did it, click here.  Press the red start button at the bottom of the Aldente page to begin the search.  The search will take a few minutes. What protein was identified?  What peptides were identified? What is the protein coverage? What masses went unidentified? What are the unidentified masses?  What is the average mass accuracy of this mass spectrometer?  Were any modifications identified?  Why do you think we missed finding some of the peptides?  If you would like to see our results page, click here.

See references 10-11 for more information on the Aldente search engine. Be sure to cite Aldente if you use any future results in a publication. How to cite

Now that you have mastered peptide-mass fingerprinting try your hand at a technique called "Sequence Tag" on the following page.




  1. Henzel, W. J.; Stults, J. T.; Watanabe, C. Proceedings of the Third Symposium of the Protein Society; Seattle, WA, 1989.

  2. Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C.  Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.  Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5011-5.

  3. Mann M, Hojrup P, Roepstorff P. Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol Mass Spectrom. 1993 Jun;22(6):338-45.

  4. Pappin DJ, Hojrup P, Bleasby AJ. Rapid identification of proteins by peptide-mass fingerprinting.
    Curr Biol. 1993 Jun 1;3(6):327-32.  (first to coin the term)

  5. James P, Quadroni M, Carafoli E, Gonnet G. Protein identification by mass profile fingerprinting.
    Biochem Biophys Res Commun. 1993 Aug 31;195(1):58-64.

  6. Yates JR 3rd, Speicher S, Griffin PR, Hunkapiller T. Peptide mass maps: a highly informative approach to protein identification. Anal Biochem. 1993 Nov 1;214(2):397-408.

  7. Jensen ON, Podtelejnikov AV, Mann M.  Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching. Anal Chem. 1997 Dec 1;69(23):4741-50.

  8. Clauser KR, Baker P, Burlingame AL.Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.
    Anal Chem. 1999 Jul 15;71(14):2871-82. PMID: 10424174

  9. Henzel WJ, Watanabe C, Stults JT. Protein identification: the origins of peptide mass fingerprinting.  J Am Soc Mass Spectrom. 2003 Sep;14(9):931-42.

  10. Tuloup M, Hernandez C, Coro I, Hoogland C, Binz P-A, Appel R D, Aldente and BioGraph: An improved peptide mass fingerprinting protein identification environment, Swiss Proteomics Society 2003 Congress: Understanding Biological Systems through Proteomics, Basel, Switzerland, 2-4 Dec. 2003, Ed. FontisMedia (ISBN 2-88476-004-0), 174-176
  11. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M R, Appel R D, Bairoch A, Protein Identification and Analysis Tools on the ExPASy Server, in: The Proteomics Protocols Handbook (in press, 2005 Feb.). Edited by John M. Walker, Humana Press, New Jersey.




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