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Quantitative Characterization of Post-Translational Protein Modifications Using Mass Spectrometry

Weijun Qian, Principal Investigator

The accurate mass and time (AMT) tag strategy.
The accurate mass and time (AMT) tag strategy. Protein samples are prepared and digested with trypsin to generate peptide mixtures. Complex samples may be subjected to peptide fractionation after digestion. The fractionated or unfractionated peptide mixture is then analyzed by LC-MS/MS resulting in spectra that are analyzed using SEQUEST for identification of the peptide. An AMT tag database containing MS/MS peptide identifications, accurate elution times, and calculated masses is generated. The AMT tag database provides a look-up table for subsequent high-throughput LC-FTICR analyses. Peptides are identified from LC-FTICR peaks when both the measured accurate masses and normalized elution times match to the ones in the AMT tag database within specified values.

Post-translational protein modifications such as phosphorylation and oxidative modifications play essential roles in the regulation of a variety of biological processes. Increasing evidence suggests that changes in protein modifications over time correlate with particular phenotypes and disease states. However, quantitative characterization of protein modifications still presents a tremendous challenge for current analytical technologies because of the extremely low abundance of modified proteins among very complex proteome samples.

The Quantitative Characterization of Post-Translational Protein Modifications Using Mass Spectrometry project at Pacific Northwest National Laboratory (PNNL) is directed toward developing global proteomic approaches for more sensitive and comprehensive characterization of modified proteins. Specifically, we will focus on the development of a quantitative proteomic approach for analyzing protein phosphorylation and oxidative protein modifications, including tyrosine nitration and methionine oxidation.

Specific enrichment methodologies for these modified peptides will be one of the keys to the success of this project. Several enrichment strategies will be developed and refined, including immobilized metal-ion affinity chromatography (IMAC), chemical enrichment, and enzyme- or antibody-based approaches. Peptide sequences and sites of modifications from the enriched samples will be identified by capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These enrichment strategies will be combined with 16 O/ 18 O stable isotope labeling and LC-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to provide the framework for quantitative characterization of modification changes in both single-sample and time-course experiments. The temporal changes in protein phosphorylation and oxidation in cells or tissues involved in specific signaling pathways will be quantitatively characterized to gain a novel understanding of the biological regulation as well as to demonstrate the capability of these approaches.

Systems Biology at PNNL

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Spatial and Temporal Proteomics Projects

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