[Proteome analysis: the state of the art of the methodology].
The proteome is the protein complement of a genome. Proteome analysis has been progressing worldwide. Two-dimensional electrophoresis (2-DE), a key technique in proteome analysis, separates proteins on a polyacrylamide gel according to the isoelectric point and molecular mass. A total of 1,000-1,500 protein spots can be separated and detected on a polyacrylamide gel using silver-staining. It is important to identify individual protein spots in order to correlate the information of the genome to that of the corresponding proteome. By automatic amino-terminal sequencing, around 15 amino acid residues from the amino terminus can be determined from 1 pmole of a protein sample. The homology search of the obtained sequences against a protein sequence database can clarify the protein unambigously. Recently, a carboxyl-terminal sequencing method using a vapor from a high concentration of an organic acid has been developed. Peptide-mass-fingerprinting is a new method for protein identification using residue-specific proteases and mass spectrometry. Two types of chemical cleavage methods, the carboxyl side cleavage of the aspartyl peptide bond and the amino terminal cleavage of serine/threonine peptide bonds would be more suitable for peptide-mass-fingerprinting of micro amount protein because of no contamination from the gel matrix or the enzyme used. It would be possible to analyse less amount protein sample (100 femtomole) more rapidly according to advancement of mass spectrometry.
Tsugita
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Research Institute for Biological Sciences, Science University of Tokyo, Chiba, Japan.
Kawakami
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D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D020543:Proteome / Q000235:genetics*
D011506:Proteins; D020543:Proteome