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Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures
Published on: September 28, 2013
Quantitative proteomics using uniform (15)N-labeling, MASCOT, and the trans-proteomic pipeline
Magnus Palmblad1, Laurence V Bindschedler, Rainer Cramer
1The BioCentre, The University of Reading, Whiteknights, Reading, UK. n.m.palmblad@reading.ac.uk
Proteomics
|August 30, 2007
Summary
This study presents an automated pipeline for analyzing uniformly labeled proteins using mass spectrometry (MS). The method efficiently processes large datasets for accurate protein quantification and identification.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Stable isotope labeling with nitrogen-14 (14N) and nitrogen-15 (15N) is crucial for quantitative proteomics.
- High-throughput techniques like LC-MS/MS generate substantial raw data requiring efficient processing.
Purpose of the Study:
- To describe a detailed approach for the automated analysis of uniformly 14N/15N-labeled proteins.
- To integrate peptide identification and abundance ratio calculation into a cohesive workflow.
Main Methods:
- Utilized stable isotope labeling (14N/15N) in conjunction with mass spectrometry (MS).
- Employed MASCOT for peptide identification and the Trans-Proteomic Pipeline (TPP) for data analysis.
- Developed scripts to integrate and automate the entire analysis workflow.
Main Results:
- Demonstrated the pipeline's effectiveness on two large proteomic datasets from Arabidopsis thaliana.
- Showcased the ability to achieve fully automated analysis from raw MS data to protein quantification.
Conclusions:
- The described automated pipeline offers an efficient and accessible solution for quantitative proteomics.
- This approach facilitates robust protein identification and abundance ratio determination using common or freely available software.
