Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug 28;135(34):12646-51.
doi: 10.1021/ja4029654. Epub 2013 Jun 4.

Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation

Jared B Shaw  1 , Wenzong Li, Dustin D Holden, Yan Zhang, Jens Griep-Raming, Ryan T Fellers, Bryan P Early, Paul M Thomas, Neil L Kelleher, Jennifer S Brodbelt
Affiliations

Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation

Jared B Shaw et al. J Am Chem Soc. .

Abstract

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods.

PubMed Disclaimer

Figures

Figure 1
Figure 1
UVPD spectra of the 11+ charge state of (A) ubiquitin and (B) the 20+ charge state of myoglobin.
Figure 2
Figure 2
Sequence coverage produced by CID, HCD, ETD and UVPD as a function of precursor ion charge state for (A) ubiquitin and (B) myoglobin. (C) UVPD product ion array for the 10+ charge state of ubiquitin showing the distribution of cleavages throughout the protein. Green and blue areas indicate the observed cleavage for N-terminal and C-terminal product ions, respectively.
Figure 3
Figure 3
Number of each product ion type observed as function of precursor ion charge state for (A) ubiquitin and (B) myoglobin.
Figure 4
Figure 4
Crystal structure (PDB ID 3NTP) of Pin1 (top left) showing the locations of C57 and C113, which are observed in non-oxidized (C57, yellow) and oxidized (C113, red) forms, respectively. Mass spectrum of intact Pin1 (top right) showing doubly and triply oxidized species as the most abundant. Zoomed-in section of a 193 nm UVPD mass spectrum (bottom) of the 22+ charge state species, deconvolved to singly protonated species. Apostrophe (’) indicates the neutral loss of water.
See this image and copyright information in PMC

References

    1. Duncan MW, Aebersold R, Caprioli RM. Nat Biotech. 2010;28:659–664. - PubMed
    1. Kelleher NL, Lin HY, Valaskovic GA, Aaserud DJ, Fridriksson EK, McLafferty FW. J. Am. Chem. Soc. 1999;121:806–812.
    1. McLafferty FW, Breuker K, Jin Mi, Han Xuemei, Infusini G, Jiang Honghai, Kong Xianglei, Begley TP. FEBS Journal. 2007;274:6256–6268. - PubMed
    1. Zhou H, Ning Z, E. Starr A, Abu-Farha M, Figeys D. Anal. Chem. 2011;84:720–734. - PubMed
    1. Little DP, Speir JP, Senko MW, O’Connor PB, McLafferty FW. Anal. Chem. 1994;66:2809–2815. - PubMed

Publication types

Substances