Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation - PubMed (original) (raw)

. 2013 Aug 28;135(34):12646-51.

doi: 10.1021/ja4029654. Epub 2013 Jun 4.

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

Jared B Shaw et al. J Am Chem Soc. 2013.

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.

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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.

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