Development of ultraviolet photodissociation for high-throughput analysis of heavily modified proteins and peptides




Greer, Sylvester McCarthy, III

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The utility of 193 nm ultraviolet photodissociation (UVPD) is evaluated for high-throughput proteomics applications including: analysis of small peptides in a traditional bottom-up proteomics workflow, analysis of heavily modified larger middle down sized peptides, and heavily modified intact proteins in a top-down proteomics workflow. UVPD uses higher energy ultraviolet photons (193 nm, 6.4 eV per photon), which are absorbed by the backbone to activate and dissociate ions effectively. UVPD dissociation is able to generate extensive backbone fragmentation enabling excellent characterization of peptides and proteins compared to traditional methods. Moreover, UVPD is also less hindered by certain experimental variables such as degree of modification, charge state and even ion polarity. These features are easily capitalized on for proteomics applications especially analysis of post translational modifications (PTM’s). Characterization of PTM’s is of great interest due to their involvement in several important cellular processes including cell signaling, tumorigenesis and gene expression. The studies covered in this work focus on utilizing the unique capabilities of UVPD to: 1.) characterize underrepresented peptides (acidic peptides and phosphopeptides) in the negative polarity including development of software for the analysis of the data generated, 2.) analyze intact proteins which have undergone extensive chemical modification and charge state augmentation, and 3.) precisely characterize histone proteins which are heavily modified due to their central role in gene expression and other transcription related functions.




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