Proteomics

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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

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Publications

UVnovo: A de Novo Sequencing Algorithm Using Single Series of Fragment Ions via Chromophore Tagging and 351 nm Ultraviolet Photodissociation Mass Spectrometry.

Robotham Scott A SA   Horton Andrew P AP   Cannon Joe R JR   Cotham Victoria C VC   Marcotte Edward M EM   Brodbelt Jennifer S JS  

Analytical chemistry 20160314 7

De novo peptide sequencing by mass spectrometry represents an important strategy for characterizing novel peptides and proteins, in which a peptide's amino acid sequence is inferred directly from the precursor peptide mass and tandem mass spectrum (MS/MS or MS(3)) fragment ions, without comparison to a reference proteome. This method is ideal for organisms or samples lacking a complete or well-annotated reference sequence set. One of the major barriers to de novo spectral interpretation arises f  ...[more]

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