Quantitative Approaches for Protein Differential Expression Analysis
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In this work, tandem mass spectrometry (MS/MS)-based quantitative protocols were developed to facilitate differential protein expression analysis and biomarker discovery via a two-step sample interrogation strategy: (a) global protein profiling and differential expression analysis by spectral counting; and, (b) biomarker candidate validation by targeted screening, i.e., multiple reaction monitoring (MRM). Preliminary experiments were performed to evaluate the performance of the spectral counting method. The method proved to be applicable for proteins with spectral countsâ ¥2, and a close-to-linear relationship between protein concentration and spectral count data was achievable at protein concentration levels <0.1 Î¼M. The detection limit was 40-800 fmol. A protein/peptide library containing ~10,000 peptide entries that facilitates the development of future MRM experiments, was developed. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum, the peptide retention time, and the top 10 most intense product ions that correspond to a given parent peptide. Only proteins identified by at least two spectral counts are listed. An MRM experiment was performed to demonstrate the successful applicability of this peptide library for the identification of putative biomarkers in proteomic samples.
- Masters Theses