NCI-III
EMSL Project ID
3163
Abstract
The first phase of this project focuses on following 5 specific milestones agreed to by the review committee.Scientific milestone 1: Demonstrate the ability to process and analyze soluble stable-isotope labeled Cys-containing polypeptides and phosphopeptides using capillary LC-FTICR from ~10,000 cells, corresponding to ~100 ng of total protein. We will also use conventional independent methods for five phosphoproteins identified using the PhIAT approach. The milestone will demonstrate the implementation of the ICAT and PhIAT labeling/isolation schemes, ability to both process small samples and then analyze the resulting polypeptide mixtures based upon work with the currently implemented version of the capillary LC-FTICR technology.
Scientific milestone 2: Make measurements for >2,000 ICAT labeled polypeptides from a single capillary LC-FTICR of a global tryptic digest derived from a stable-isotope labeled sample containing ~100 ng of total protein.
Scientific milestone 3: Demonstrate the use of peptide elution time information for effective “data-directed” multiplexed-MS/MS of peptides in two consecutive capillary LC-FTICR runs. We will also demonstrate the ability to use these “data-directed” multiplexed-MS/MS analyses to identify peptides having distinctive abundance ratios. We will demonstrate the identification of >100 labeled polypeptides that are computer selected on the basis of the abundance ratios for the two stable-isotope labeled versions of the peptide measured in a previous run for a duplicate sample.
Scientific milestone 4: Demonstrate that the capillary LC-FTICR approach is sufficiently sensitive to detect known proteins that are added to cell extracts (and which are not initially present) at a level of 10 pgram/100 ngran of total protein.
Scientific milestone 5: Demonstrate that the PhIAT chemistry is sufficiently robust to detect 2-fold concentration changes in a foreign protein (e.g. casein) that is added to cell extracts at a level of 0.1% and 0.05% of 100 ngran of total protein mass.
Project Details
Project type
Exploratory Research
Start Date
2002-12-05
End Date
2003-09-29
Status
Closed
Released Data Link
Team
Principal Investigator