Differential Isotopic Esterification for Quantitative Proteome Analysis and de novo Sequenceing
EMSL Project ID
2108
Abstract
The proposed method uses differential, isotopic per methyl-esterification of carboxylic acids in peptides as a method for relative quantitation of proteins between solutions of identical proteins that differ only in the relative concentration of the identical proteins. Proteins are digested to peptides and esterified using either d0- or d3-methanol. The peptides from different states modified differentially with either d0- or d3-methanol are then mixed and analyzed by microcapillary HPLC-ESI/MS/MS. This chemical labeling scheme provides each peptide with an internal standard for normalization and thus relative quantitation. de novo sequencing is also possible using the same data set used for quantitation by virtue of the fact that sets of tandem mass spectra for identical peptides are acquired during analysis. The method and the process have been analyzed on an ion trap MS at ISB. The main expectation of analysis by FTMS is that the additional mass resolution will allow for better de novo sequence analysis. This will be effected by isotopic modeling of the data to clear out signals due to chemical noise using software developed at the ISB. Samples would be generated at the ISB for quantitation and de novo sequence analysis as follows. These samples would be prepared by 1) digest sperm whale myoglobin with trypsin, 2) split into aliquots equal in protein amount, 3) esterify one aliquot with d0-methanol and a second with d3-methanol, 4) mix the two, now different, myoglobin digests 2:1, 5) analyze by FTMS using protocols to fragment the peptide ion pairs in such a way that the original 2:1 ratio is maintained, and 6) interpret the peptide sequence by de novo data analysis using software being developed at the ISB.
Project Details
Project type
Exploratory Research
Start Date
2000-11-29
End Date
2002-05-01
Status
Closed
Released Data Link
Team
Principal Investigator