In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 93, No. 25 ( 1996-12-10), p. 14440-14445
Abstract:
The function of many of the uncharacterized open reading frames
discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate
gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here
that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen,
our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic
digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with
high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by
nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials,
the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of
150 gel spots—many of them at subpicomole amounts—were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More
than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that
mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of
choice to connect genome and proteome.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.93.25.14440
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
1996
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
Bookmarklink