Pregled bibliografske jedinice broj: 662370
De novo sequencing of Pleuroteus ostreatus proteins by use of isotopic lageling in combination with tandem mass spectrometry
De novo sequencing of Pleuroteus ostreatus proteins by use of isotopic lageling in combination with tandem mass spectrometry // 35th Annual Meeting of the German Society for Mass Spectrometry
Heidelberg, Njemačka, 2002. (poster, domaća recenzija, neobjavljeni rad, znanstveni)
CROSBI ID: 662370 Za ispravke kontaktirajte CROSBI podršku putem web obrasca
Naslov
De novo sequencing of Pleuroteus ostreatus proteins by use of isotopic lageling in combination with tandem mass spectrometry
Autori
Matis, Maja ; Jovanović, Marko ; Mormann, Michael ; Žakelj-Mavrič, Marija ; Peter-Katalinić, Jasna
Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, neobjavljeni rad, znanstveni
Skup
35th Annual Meeting of the German Society for Mass Spectrometry
Mjesto i datum
Heidelberg, Njemačka, 03.03.2002. - 06.03.2002
Vrsta sudjelovanja
Poster
Vrsta recenzije
Domaća recenzija
Ključne riječi
de novo sequencing; isotopic labeling; tandem MS
Sažetak
17ß-hydroxysteroid dehydrogenase (17ß-HSD) catalyzes reversible interconversions of 17-hydroxy and 17-keto steroids, substrates that play a crucial role in biosynthesis of estrogens and androgens as well as in regulation of steroid hormone activities. Because these enzymatic activities are common to higher organisms and not to microorganisms, we analyzed purified fractions from the fungi P. ostreatus positively tested for 17ß-HSD catalytic activity. Since the P. ostreatus genome has not been sequenced yet and that there is only a low degree of homology in primary structure between proteins with the 17ß-HSD enzymatic activity from different organisms, another strategy for identification has been chosen. The peptides obtained after proteolytic digestion with trypsin in H218O medium were isotopically labeled at the C-terminus prior to electrospray quadrupole time-of-flight (ESI-QTOF) tandem mass spectrometry analysis. Using this strategy we obtained clear-cut peptide fragmentation spectra - manually interpretable - showing full sequence data from appropriate b- and y-ions. This aspect seems to be crucial for de novo sequencing of unknown proteins. With the peptide sequence data obtained by MS/MS we searched the protein databases with FASTA and BLAST programs. In this way we identified a protein with a molecular weight of 36 kDa, expressing high homology (60 to 70%) with E. coli malate dehydrogenase. To identify other proteins we are using polymerase chain reaction (PCR) technique to get more information on the primary structure of proteins that have lower or no homology with other proteins. On the basis of the larger part of the sequence we intend to identify the protein(s) showing 17ß-HSD catalytic activity.
Izvorni jezik
Engleski
Znanstvena područja
Kemija, Biologija, Biotehnologija
