Proposal title: Characterization of structural features of modified rhm-CSF b by using mass spectrometric techniques
EMSL Project ID
2066
Abstract
Project description: Recombinant human macrophage colony stimulating factor (rhm-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of monocytes and macrophases. It is potentially useful in treating infection and malignancies, also in accelerating the hemopoietic recovery following chemotherapy or bone marrow transplant. The biologically active rhm-CSF b exists as a homodimer, linked by nine inter- and intramolecular disulfide bonds, and provides an interesting and challenging system for studying the roles of disulfide bonds in determining protein conformation. Researchers of Max Deinzer's laboratory at Oregon state university have been exploring the possibility of using cyanylation to block sulfhydryl groups during oxidative in vitro refolding of rhm-CSF b. The practical question to be answered in this project is how many cyanylations occur to the protein among the 18 available cystein residues and where specifically the cyanylation to occur. Preliminary experiments indicated two cyanylations occurred to the protein upon partial reduction of disulfide bonds and subsequent cyanylation. We propose to use FT-ICR to measure accurate mass of the modified proteins. It was shown that FT-ICR feasibly measure masses of large biological polymers within a few ppm level. This would give direct answer to the first question of this project which is the number of cyanylation, with comparison to its native protein. To investigate the site of cyanylations, we propose multiplex MS/MS experiments using FT-ICR on tryptic digest samples of the modified proteins.
Project Details
Project type
Exploratory Research
Start Date
2000-09-20
End Date
2002-02-28
Status
Closed
Released Data Link
Team
Principal Investigator