Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

Abstract

Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

Ion Activation Methods for Peptides and Proteins

“…244 Low energy collisional activation provides insight into the sub-unit composition of the complexes (stoichiometry) but not sequence information about the individual proteins. 244 Other ion activation methods, including higher kinetic energy beam type CID 245 and higher energy collisional dissociation (HCD), [246][247] surface induced dissociation (SID), [138][139][140][141][142][143][144] electron capture dissociation (ECD), [248][249][250][251][252][253][254] electron transfer dissociation (ETD), [255][256][257] and photodissociation, [258][259][260][261][262][263] have also been used to evaluate structures of native proteins and protein complexes.…”

“…259 193 nm UVPD has been used to characterize the sequences and structures of native proteins and protein-ligand complexes, as well as map the binding sites of the ligands. [260][261][262][263] Variations in the efficiencies of backbone cleavages reflected the interactions of ligands with the protein, either via suppressing fragmentation from the formation of stabilizing interactions between the ligand and protein or by enhancing fragmentation owing to disruption of stabilizing non-covalent interactions. Using this UVPD strategy, complexes of myoglobin/ heme, 261-262 eIF4E/m 7 GTP, 261 and peptidyl-prolyl cis-trans isomerase 1/C-terminal domain of RNA polymerase II (CTD) peptide 261 were examined.…”

Structural characterization of holo- and apo-myoglobin in the gas phase by ultraviolet photodissociation mass spectrometry

“… 4 This process can be simplified by adoption of a top-down workflow which alleviates the proteolytic digestion step but requires a high-resolution/high mass accuracy mass spectrometer to map the incorporation of the covalent labels. 5 , 6 For the top down approach, the deuterium-labelled or probe modified proteins are typically transferred to the gas phase by electrospray ionization (ESI) and fragmented by collisional induced dissociation (CID) or its analog higher energy collisional dissociation (HCD), electron-based activation (ECD or ETD), or ultraviolet photodissociation (UVPD), then the modified sites are determined based on interpretation of the abundances of resulting fragment ions. 5 , 6 Denaturing ESI conditions are typically used to maximize sensitivity.…”

Ultraviolet photodissociation of protonated, fixed charge, and charge-reduced peptides

“…"Hybrid" activation methods combine electron transfer reactions with CAD, higher-energy collisional dissociation (HCD), infrared multiphoton dissociation (IRMPD) or UVPD and have been reported to circumvent some of the potential charge-dependent shortcomings of ECD or ETD alone to generate richer fragmentation patterns [38,39,[44][45][46][47][48][49][50][51]. UVPD has been previously shown to produce rich fragmentation patterns alone compared to other methods [52,[55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72]. We have previously used 193 nm UVPD to explore the fragmentation of radical peptide cations generated by ETnoD [52].…”