Xenoestrogen effect on trout histones
EMSL Project ID
38512
Abstract
We are using a fish model, the rainbow trout, as a human surrogate to investigate the reproductive consequences of xenoestrogen exposure on male reproduction. Our previous studies have documented significantly reduced embryo survival resulting from fathers that were exposed to the xenoestrogen 17(alpha)-ethynylestradiol and sperm aneuploidy. These results provide evidence for a novel mechanism in which xenoestrogens affect the survival of the next generation. We hypothesize that paternal genome packaging involving somatic histones could be responsible. Preliminary studies, in collaboration with the PNNL Environmental Molecular Sciences Laboratory (EMSL), has shown that histones can be recovered from a trout testes sample for proteomic analysis. An in vivo experiment has been conducted in which male rainbow trout were exposed to EE2 and total histone isolations have been prepared. This General grant proposal seeks a comparative histone analysis using EMSL proteomic capabilities. Comparisons of the number of histones and variations in their quantity between the two treatment groups will provide the necessary preliminary data for a planned National Institutes of Health (NIH) grant submission. We will utilize 12T FTICR MS to obtain LC-MS data to be used for quantitative label-free profiling of histones isolated from testis samples. Differentially abundant proteins will be incorporated into a target ion list for subsequent offline MS/MS using 12T FTICR with collisionally induced dissociation (CID) and electron capture dissociation (ECD) and an aliquot of the collected protein fraction. We will use publicly available and PNNL-developed tools for alignment of LC-MS datasets, label-free (MS-peak intensity) based quantitation and MS/MS-based intact protein identification. We will also use intact protein masses in conjunction with bottom-up data obtained for the same LC fraction to tentatively identify all of the detected species and protein MS/MS for follow-up characterization of interesting targets. For bottom-up analysis, LC fractions collected during the LC-MS analyses will be tryptically digested; resulting peptide mixtures will undergo LC-MS/MS analysis coupled with an ion trap to alternatively acquire CID and ETD spectra (e.g. LTQ-ETD). The resulting MS/MS data (peptide and protein level) will be analyzed against published sequences for trout histones and protamines and multiple protein databases (MSDB, PubMed) using commercial and PNNL-developed computational tools. This EMSL General proposal would be used to obtain preliminary data to support a proteomic objective in a future NIH grant proposal and justify inclusion of EMSL in the NIH proposal work plan.
Project Details
Project type
Exploratory Research
Start Date
2010-03-24
End Date
2011-03-27
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members