193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology

Abstract

Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins.

Figure 1

HCD and UVPD of 13+ and 15+ tetrameric streptavidin. In a) and d) HCD of the 13+ and 15+ charge states is shown, respectively. In b) and e) 1.0 mJ UVPD of the 13+ and 15+ charge states is shown and in c) and f) 3.0 mJ UVPD of the 13+ and 15+ charge states is shown. The bracket in c) denotes the region populated by fragments originating from cleavages of the protein backbone (i.e. sequence-type ions).

Figure 2

UVPD ERMS plots of the a) 13+ and b) 15+ charge states of tetrameric streptavidin. High charged monomers included charge states 6+ and higher, and low charged monomers included charge states 5+ and lower.

Figure 3

HCD and UVPD of tetrameric transthyretin (15+); in a) HCD (50 eV) is shown, in b) 1.0 mJ UVPD is shown, and in c) 3.0 mJ UVPD is shown. UVPD ERMS plot of tetrameric transthyretin (15+) is shown in d) and the weighted average charge states of the trimer and monomer products upon UVPD are shown in e). In d), high charged monomer includes charge states 7+ and higher, and low charged monomer includes charge states below 7+.

Figure 4

HCD (a), 1 mJ UVPD (b), and 3 mJ UVPD (c) mass spectra of the 17+ charge state of tetrameric HG. The relative abundance of holo monomers are shown in d) as a function of laser power. In e) a UVPD-ERMS plot is shown for the production of high charged (8+ to 10+) monomers, low charged monomers (4+ to 7+), and backbone cleavage products (sequence ions) of HG (17+) as a function of laser power. Trimer ions are not shown and (subunit) dimers were not observed as products.

Figure 5

For streptavidin: a) B-factor values and solvent accessible surface areas (SASA), b) UVPD fragmentation yields (15+) and c) the crystal structure of SA (pdb 1SWB) is highlighted such that regions featuring enhanced UVPD fragmentation are shown in red. In b), the UVPD fragmentation yield of the 6+ monomer of SA, generated from source CID, is shown in green. For transthyretin: d) B-factor values and solvent accessible surface areas (SASA), e) UVPD fragmentation yields (15+), and f) the crystal structure of TTR (pdb 4TLT) is highlighted such that regions featuring enhanced UVPD fragmentation are shown in red.

Figure 6

SASA values, B-factors, and UVPD fragmentation yields are shown for alpha (a–c) and beta (d–f) hemoglobin. B-factor is plotted in blue and SASA in red in a) and d). Fragmentation yield following 3.0 mJ UVPD of the 17+ charge state is shown in for alpha HG (b) and beta HG (e). In c) and f), the crystal structures of alpha and beta hemoglobin (pdb 1BBB) are highlighted such that regions featuring enhanced UVPD fragmentation are shown in red. Cleavages C-terminal to Phe or Tyr are denoted F| and Y|. Cleavages N-terminal to Pro are denoted |P.