Ground squirrel proteome: protein modifications through torpor and arousal
EMSL Project ID
25671
Abstract
Liver samples from the hibernator, golden-mantled ground squirrel, when separated by 2D gel electrophoresis, demonstrate a quantifiable and as yet undefined physical modification that manifests itself as fuzzy protein spots. All evidence suggests that this subtle modification accumulates during the time that the animal is in torpor and reverses upon winter arousal as a normal part of the hibernation cycle. EMSL high performance mass spectrometry may enable determination of both the site of this modification and its chemical identity. We postulate that the modification is disabling to protein function, and that the reversal of its accumulation may be essential for surviving hibernation. This natural ability to reverse such damage may also have broader implications for other systems in which deleterious protein adducts accumulate, such as in aging and diabetes.Initially, a liver sample from an Entrance animal (sacrificed while entering into torpor, body temp decreasing from 37°C to 5°C) will be compared to a liver sample from a Late Torpor animal (sacrificed after several days in torpor, body temp ~5°C), with the expectation that the sample size will be increased after establishing the methods. We expect to see the differences in protein modifications most clearly between these two groups. Samples will be separated using the TriVersa system in combination with RPLC-FTICR for precise intact protein mass determination correlated to the relevant fraction. The original unfractionated samples and selected fractions will be analyzed after digestion with trypsin to identify peptides/proteins and also as intact proteins to identify and locate any modifications. Peptide level analysis will be performed using high resolution LC-MS/MS (i.e. ThermoElectron Finnigan LTQ-Orbitrap) in an attempt to identify both bare and modified peptides, while protein level MS/MS will use an FTICR mass spectrometer with ECD and CID capabilities. Comparison of data from the two samples will allow for tentative matching of modifications with protein identifications and reveal any substantial differences that develop during torpor on these proteins.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-06-26
End Date
2008-06-29
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members