FEMTOMOLE SEQUENCING OF PROTEINS FROM POLYACRYLAMIDE GELS BY NANO-ELECTROSPRAY MASS-SPECTROMETRY

Citation
M. Wilm et al., FEMTOMOLE SEQUENCING OF PROTEINS FROM POLYACRYLAMIDE GELS BY NANO-ELECTROSPRAY MASS-SPECTROMETRY, Nature, 379(6564), 1996, pp. 466-469
Citations number
16
Categorie Soggetti
Multidisciplinary Sciences
Journal title
NatureACNP
ISSN journal
00280836
Volume
379
Issue
6564
Year of publication
1996
Pages
466 - 469
Database
ISI
SICI code
0028-0836(1996)379:6564<466:FSOPFP>2.0.ZU;2-#
Abstract
MOLECULAR analysis of complex biological structures and processes incr easingly requires sensitive methods far protein sequencing. Electrospr ay mass spectrometry(1) has been applied to the high-sensitivity seque ncing of short peptides(2), but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple an d robust technique for the sequencing of proteins isolated by polyacry lamide gel electrophoresis, using nano-electrospray(3,4) tandem mass s pectrometry(5,6). As little as 5 ng protein starting material on Cooma ssie- or silver-stained gels can be sequenced. Multiple-sequence stret ches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information t o clone the corresponding gene. We have applied this method to the seq uencing and cloning of a protein which inhibits the proliferation of c apillary endothelial cells in vitro and thus may have potential antian giogenic effects on solid tumours.