Protein Characterization: Analytical Approaches and Applications to Proteomics

Abstract

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: phenylisothiocyanate; PTC: phenylthiocarbamyl; PTH: phenylthiohydantoin.

Figure 2.

Example of an MALDI‐TOF‐MS 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 TOFMS/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.

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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.

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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

Matrix Science http://www.matrixscience.com

BLAST http://www.ncbi.nih.gov/BLAST

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