De Novo Sequencing of Peptides Using Selective 351 nm Ultraviolet Photodissociation Mass Spectrometry

peer-reviewed · Analytical Chemistry · 2013

peer-reviewed · Analytical Chemistry · 2013. Scott A. Robotham et al. Although in silico database search methods remain more popular for shotgun proteomics methods, de novo…
Date 2013-10-15
Type peer-reviewed
Venue Analytical Chemistry
Publisher American Chemical Society (ACS)
Contribution adjacent
DOI 10.1021/ac402309h
Citations (OpenAlex) 14
Venue 2-year citedness 7.10

Abstract

Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify peptides derived from 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 generates a characteristic series of y ions. The UVPD spectra of the chromophore-labeled peptides are simplified and thus amenable to de novo sequencing. This method resulted in an observed sequence coverage of 79% for cytochrome C (eight peptides), 47% for β-lactoglobulin (five peptides), 25% for carbonic anhydrase (six peptides), and 51% for bovine serum albumin (33 peptides). This strategy also allowed differentiation of proteins with high sequence homology as evidenced by de novo sequencing of two variants of green fluorescent protein.

Authors

  1. Scott A. Robotham · University of Texas at Austin
  2. Christien Kluwe · University of Texas at Austin
  3. Joe R. Cannon · University of Texas at Austin
  4. Andrew Ellington · University of Texas at Austin
  5. Jennifer S. Brodbelt · University of Texas at Austin

Methods and tools

  • 351 nm UVPD de novo sequencing: N-terminal chromophore derivatisation makes a peptide absorb at 351 nm, so selective UV photodissociation cleaves it into a simplified y-ion series instead of the mixed ladders CID gives, deliberately to make the spectra amenable to de novo sequencing.

Cites (28)

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