Date of Award
Dr. Richard Cole
Dr. Jonathan Lansing
Dr. Yang Cai
Dr. Mark Trudell
Positional proteomics is emerging as an attractive technique to characterize protein termini, which play important biological roles in cells. Even with the advances in past decades, there still are areas for improvement. This thesis focuses on improving data quality and assignment confidence in positional proteomics.
A novel workflow was designed for the large-scale identification of protein N-terminal sequences. 4-sulfophenyl isothiocyanate (SPITC) is used for N-termini sulfonation; Upon higher energy collisional dissociation (HCD), SPITC peptides in electrospray ionization ESI generate predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high fidelity. The presence of b1 + SPITC product ions upon HCD furthers the confidence for N-terminal identifications. Secondly, sulfonated N-terminal peptides possess one negative charge site at low pH, which was exploited to enrich the SPITC modified N-terminal peptides by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography. Such enrichment process allows both N-termini enriched and N-termini deficient fractions to be collected and analyzed by LC-MS/MS. This method was applied to an E. coli cell lysate, identifying approximately 350 N-terminal peptides (85% represented neo-N-termini from protein degradation and 15% from leading methionine excision). These N-terminal peptides represented 274 distinct E.coli proteins, 224 of which were also identified in the analysis of flow-through fractions from internal peptides.
Another approach we took to boost the identification confidence is by exploiting iTRAQ (isobaric tag for relative and absolute quantitation) in the positional proteomics workflow. This approach allows for multiplexed comparison between different samples, and thus is well-suited for degradadomics analyses where degraded samples are compared to control samples. Both control and protease treated sample are labeled by different tags which allows direct comparison of protein N-termini with neo-N-termini. In addition, samples are analyzed duplicate by labeling with two tags, aiming for quick validation of peptides by internal replicates. In this study, Asp-N digested E.coli cell lysate is taken as a model system. A total of 500 N-terminal peptides, corresponding to 370 proteins, were identified with high confidence in one experiment, with 87% of those proteolytic products matching the expected protease digestion specificity, validating the assignment accuracy of this approach.
Jiang, Yanjie, "Approaches for Improved Positional Proteomics" (2013). University of New Orleans Theses and Dissertations. 1715.