Peptide sequencing by mass spectrometry for homology searches and cloning of genes.
It is now possible to obtain sequence information from gel-separated proteins by mass spectrometry at levels too low for conventional approaches. Usually this tandem mass spectrometric data are used for database searches with the aim of identifying the corresponding gene. Recently it has been shown that long and accurate amino acid sequences can be obtained which are sufficient for PCR-based strategies to clone the corresponding gene [Wilm et al. (1996), Nature 379, 466-469]. More than eight proteins have now been cloned based on that method. In many more cases the sequence information identified homologous proteins. Issues involved in cloning by mass spectrometric sequence information are discussed, as are two case studies. These results clearly establish mass spectrometry as a viable tool not only for the database identification of proteins, but also for the de novo sequencing of gel-separated proteins at the low-picomole to femtomole level.
Shevchenko
A
A
Protein & Peptide Group, European Molecular Biology Laboratory, Heidelberg, Germany.
Wilm
M
M
Mann
M
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D010455:Peptides; D004247:DNA