Quantifying Peptide Abundance Ratios
EMSL Project ID
5112
Abstract
Regulation of protein expression and modulation of post-translational modifications (PTMs) are biochemical processes that are vital to cellular function. Phosphorylation and glycosylation are two of the more important PTMs in eukaryotic cells since they are involved in many cell signaling cascades and pathways. Various methods using stable isotope labeling have been reported to examine global protein expression levels, but few have the ability to quantify and monitor PTMs. A combination of metabolic labeling using 14N/15N isotopically enriched media and the phosphoprotein isotope-coded affinity tag (PhIAT) approach using LC-MS/MS has been developed to identify and quantify of phosphorylation and glycosylation for global proteomic studies.The use of 14N- or 15N-enriched media permitted the isotope labeling of proteins of yeast exposed to various carbon sources to occur at the transcriptional level. In this manner, all the proteins expressed under a given set of growth conditions were isotopically labeled. For characterizing protein expression, part of the sample was digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The remaining protein fraction was used to assess changes in phosphorylation/ glycosylation by simultaneously PhIAT labeling the pSer/pThr and O-glycosylated residues. The PTMs at these residues were removed by hydroxide ion-mediated beta-elimination then labeled with ethanedithiol via Michael addition and subsequently biotinylated. The PhIAT-labeled peptides were analyzed by LC-MS/MS. In both cases, the 14N/15N labeled peptides were analyzed by LC-MS/MS using a 7-T FTICR mass spectrometer as part of a previous collaboration within EMSL Peptide identification of the product ions produced by SORI CID were analyzed using IDL-designed software (ICR-2LS).
This proposal is directed towards continuing the analysis of these datasets in order to identify the changes occurring in yeast protein abundance and the phosphorylation/glycosylation states of the detected proteins. In this collaboration with IDL, the 14N/15N peptide abundance ratios from the FTICR data we have previously collected and were archived in the DMS system at EMSL will be determined using IDL developed software specifically designed for this purpose. All the runs were SORI CID acquisitions ? an MS scan followed by a multiplex MS/MS scan; we did not perform a MS only acquisition for our samples. Consequently, the duty cycle employed during ICR analysis could be problematic for abundance ratio measurements. However, since we used an LC system with formic acid as the ion-pairing agent, there should not be any detectable differences in retention time between the 14N/15N peptide pairs. Each pair will co-elute and their relative abundance ratios will be maintained during any point in the MS acquisition and thus permit quantitation. The data obtained from this study will be part of a manuscript to be submitted for publication with the appropriate IDL personal involved in the data analysis listed as co-authors.
Project Details
Project type
Exploratory Research
Start Date
2003-10-29
End Date
2005-11-11
Status
Closed
Released Data Link
Team
Principal Investigator
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