De novo sequencing and peptide characterization via ultraviolet photodissociation mass spectrometry




Robotham, Scott Allen

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Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generated characteristic series of y ions. The UVPD spectra of the chromophore-labelled peptides were simplified and thus amenable to de novo sequencing. E.coli lysates were modified by the use of carbamylation and the attachment of a UV chromophore to the N-terminus of digested peptides. UVPD of the resulting peptides generated clean sets of y ions. A novel de novo algorithm, UVnovo, afforded high confidence identification of thousands of peptides from an E. coli lysate and allowed UVPD/CID paired spectra to be searched. E.coli lysate peptides were analyzed in alternating scans by UVPD and CID on the same precursors to generate paired UVPD/CID spectra. UVnovo enabled sequence tag de novo sequencing of peptides in order to find matching sequences from a database. Ultimately the UVnovo functioned as a standalone de novo sequencing program or a hybrid de novo/database search program. In an effort to better characterize the fragmentation pathways promoted by ultraviolet photoexcitation in comparison to CID, six adrenocorticotropic hormone (ACTH) peptides in a range of charge states were subjected to 266 nm ultraviolet photodissociation (UVPD), 193 nm UVPD, and CID. While both UVPD and CID led to preferential cleavage of the Y-S bond for all ACTH peptides (except ACTH (1-39)), UVPD was far less dependent on charge state and location of basic sites for the production of C-terminal and N-terminal ions.



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