AMCA chromophore tagging and 351 nm ultraviolet photodissociation enables de novo sequencing of E. coli proteome

ABSTRACT: Demonstration of novel experimental and computational pipeline for high performance de novo peptide sequencing. E. coli whole cell lysate is carbamylated to block lysine side chains, digested with trypsin (now active only at Arg residues), and N-terminally tagged with the chromophore AMCA. Three technical replicates were analyzed using a Thermo Velos Pro dual linear ion trap mass spectrometer coupled to a Coherent 351 nm excimer laser. 351 nm ultraviolet photodissociation (UVPD) of parent peptides produces MS2 spectra dominated by the y-type ion series. We developed the software tool UVnovo for de novo sequencing of these spectra and used Proteome Discoverer SEQUEST/Percolator to generate a dataset for training and validation. UVnovo results provided here derive from a 3-fold cross validation regime. Our methods and dataset are described in the accompanying publication.

INSTRUMENT(S): Thermo Scientific Velos Pro mass spectrometer modified to enable 351 nm UVPD

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Jennifer Brodbelt, Edward Marcotte 

PROVIDER: MSV000079571 | MassIVE | Thu Mar 10 19:28:00 GMT 2016

SECONDARY ACCESSION(S): PXD003767

REPOSITORIES: MassIVE