Protein Characterization: Analytical Approaches and Applications to Proteomics


Edman chemical sequencing and biological mass spectrometry are used routinely to identify and to characterize proteins. Two‐dimensional polyacrylamide gel electrophoresis, highly sensitive mass spectrometry and sequence database searching algorithms have merged to form proteomics – the analysis of the protein complement of the genome.

Keywords: mass spectrometry; proteomics; protein identification; Edman degradation; bioinformatics

Figure 1.

The different steps involved in chemical Edman sequencing. ATZ: anilinothiazolinone; PITC: ; PTC: ; PTH: phenylthiohydantoin.

Figure 2.

Example of an MALDITOFMS peptide mass fingerprint obtained from a tryptic digest of Escherichia coli malate dehydrogenase isolated by 2D‐PAGE. Tryptic peptides belonging to the identified protein are indicated by their masses. T indicates trypsin autodigestion products.

Figure 3.

Quadrupole TOF MS/MS spectrum of a peptide obtained after RP‐HPLC separation of the tryptic digestion of an affinity‐isolated protein. The doubly charged ion of a peptide with mass 1351.63 Da was selected automatically by the mass spectrometer and fragmented by collision‐activated dissociation. The obtained MS/MS spectrum was used automatically for peptide identification by the Mascot search algorithm (Matrix Science). The amino acid sequence NH2‐T‐A‐S‐G‐S‐S‐V‐T‐S‐L‐E‐G‐T‐R‐COOH (in single‐letter code) was returned as the best matching peptide and belongs to the n‐myc downstream regulated gene 1 protein (GenBank accession number Q62433). After automatic identification, the MS/MS spectrum was scanned manually for the presence of y‐type fragment ions, through which a large portion of the proposed amino acid sequence could be deduced.



Biemann K (1988) Contributions of mass spectrometry to peptide and protein structure. Biomedical and Environmental Mass Spectrometry 16: 99–111.

Cottrell JS (1994) Protein identification by peptide mass fingerprinting. Peptide Research 7: 115–124.

Edman P (1949) A method for the determination of the amino acid sequence in peptides. Archives of Biochemistry and Biophysics 22: 475–480.

Eng JK, McCormack AL and Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 11: 976–989.

Fenn JB, Mann M, Meng CK, Wong SF and Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246: 64–71.

Hewick RM, Hunkapiller MW, Hood LE and Dreyer J (1981) A gas–liquid–solid‐phase peptide and protein sequencer. Journal of Biological Chemistry 256: 7990–7997.

Hunt DF, Yates III JR, Shabanowitz J, Winston S and Hauer CR (1986) Protein sequencing by tandem mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America 83: 6233–6237.

Karas M and Hillenkamp F (1988) Laser desorption/ionization of proteins with molecular masses exceeding 10000 Da. Analytical Chemistry 60: 2299–2301.

Mann M and Wilm M (1994) Error‐tolerant identification of peptides in sequence databases by peptide sequence tags. Analytical Chemistry 66: 4390–4399.

Wilm M and Mann M (1996) Analytical properties of the nano‐electrospray ion source. Analytical Chemistry 68: 1–8.

Further Reading

Gevaert K and Vandekerckhove J (2000) Protein identification methods in proteomics. Electrophoresis 21: 1145–1155.

Godovac‐Zimmermann J and Brown LR (2001) Perspectives for mass spectrometry and functional proteomics. Mass Spectrometry Reviews 20: 1–57.

Griffiths WJ, Jonsson AP, Liu S, Rai Dk and Wang Y (2001) Electrospray and tandem mass spectrometry in biochemistry. Biochemical Journal 355: 545–561.

Jungblut P and Thiede B (1997) Protein identification from 2‐DE gels by MALDI mass spectrometry. Mass Spectrometry Reviews 16: 145–162.

Kellner R, Lottspeich F and Meyer HE (1999) Microcharacterization of Proteins. Weinheim: Wiley‐VCH Verlag GmbH.

Neubauer G, King A, Rappsilber J, et al. (1998) Mass spectrometry and EST‐database searching allows characterization of the multi‐protein spliceosome complex. Nature Genetics 20: 46–50.

Shively JE (2000) The chemistry of protein sequence analysis. In: Jollès P and Jörnvall H (eds.) Proteomics in Functional Genomics, pp. 99–117. Basel: Birkhäuser Verlag.

Siuzdak G (1996) Mass Spectrometry for Biotechnology. San Diego, CA: Academic Press.

Yates JR III (1998) Mass spectrometry and the age of the proteome. Journal of Mass Spectrometry 33: 1–19.

Web Links

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Gevaert, Kris, and Vandekerckhove, Joël(Jan 2006) Protein Characterization: Analytical Approaches and Applications to Proteomics. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0005725]