This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. H-Ras is a plasma membrane associated GTPase, which is an important player in signal transduction to control cell proliferation, differentiation, and invasion. There are four reactive cysteines (C118, C181, C184 and C186) on H-Ras. These cysteines are targets of posttranslational modifications (PTM) which may alter the cellular localization and function of this protein. In this study, PTMs on H-Ras in human aortic endothelial cells (HAECs) is being investigated by mass spectrometry to obtain the full map of H-Ras modifications under different oxidative stresses. H-Ras is not an abundant protein that can be easily extracted from cell lines. A protocol has been developed to facilitate the enrichment and purification of the H-Ras, where exogenous H-Ras with 6X His tag was transduced by adenovirus, and pulled down by magnetic Ni-NTA beads. Tryptic in-gel digestion was performed on the His-tagged H-Ras, followed by MS analysis using both MALDI-TOF and LTQ-Orbitrap. Although high sequence coverage was obtained by combining these two methods, only C181 and C184 containing peptides were identified in the mass spectrum. The C186 containing tryptic peptide CVLS was absent in either experiment, because VLS is known to be cleaved after C186 gets farnesylated. Since two cysteine residues of interest were not covered in the bottom-up analysis, a top-down approach by Fourier-transform ion cyclotron resonance (FT-ICR) MS is now being developed. Several methods have been tried for the purification of the H-Ras sample, including those that use the Ziptip (C4 and C18) or centrifugal filters, dialysis, and the CapLC. It was found that the combination of dialysis and use of the Poros 50 R1 column provides the best result with the highest protein recovery yield and least interference from polyethylene glycol (PEG) and other undesirable contaminants. HPLC and Michrom desalting trap will be tried next.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-15
Application #
8365567
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Project Start
2011-06-01
Project End
2012-08-09
Budget Start
2011-06-01
Budget End
2012-08-31
Support Year
15
Fiscal Year
2011
Total Cost
$18,464
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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