[Blueboard] Seminar Invitation

mtandang at ateneo.edu mtandang at ateneo.edu
Wed Feb 9 15:31:47 PHT 2005

We invite you to attend a


(Micropterus salmoides)" by Ms. Ma. Cristina Dancel of the Department of
Chemistry,University of Florida this February 21, 2005 (Monday),
4:30-6:00 pm at Schmitt Hall CH-109.


Novel Aspect:
Tissue-specific distribution of three estrogen receptor isotypes in
Micropterus salmoides

The discovery of estrogen receptor (ER) alpha and beta in humans and other
species has helped to understand tissue-specific effects of estrogen and
estrogenic chemicals.  In our group, we believe that the presence of
three ER mRNA in largemouth bass (Micropterus salmoides) indicates the
presence of three different isotypes: ER-alpha, ER-beta and ER-gamma.  To
verify this, we are developing detection methods that use tandem mass
spectrometry to determine the levels of these isotypes in various
largemouth bass tissue extracts.

We currently use proteomics to identify characteristic peptides for the
largemouth bass ERs.  One of these peptides will be synthesized with
heavy isotopes and will be used in the analysis of tissue extracts. 
Initial experiments involve method development and validation by in-gel
proteolytic digestion of recombinant ER-alpha ligand-binding domain
(LBD), the region where the three ER isotypes have ~50% sequence
homology.  MALDI MS is used to analyze the resulting peptide mixture
followed by database search and sequence alignment.

Preliminary Results:
In-gel trypsin digestion followed by MALDI-TOF MS identified a number of
peptides equivalent to 54% coverage of the ER-alpha LBD.  At least seven
peptides have medium to strong ion intensities.  Two of these are unique
to the recombinant protein, while the rest are also found in ER alphas
from other fish species.  We have chosen one of the latter, the peptide
LIFAQDLILDR, for our quantification experiments.  Meanwhile, in silico
trypsin digests of the other two isotypes did not show any of the
peptides observed with ER-alpha LBD.  Recombinant expression of the
full-length ER-alpha, as well as ER-beta and ER-gamma, are underway.

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