Native electrospray and electron-capture dissociation in FTICR mass spectrometry provide top-down sequencing of a protein component in an intact protein assembly

Abstract

The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23ϩ to 27ϩ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment. (J Am Soc Mass Spectrom 2010, 21, 1966 -1968 ECD and FT (Fourier transform) ion cyclotron resonance (ICR) MS combine as an appealing approach for study of protein assemblies because, in principle, the masses of the complex and of the subunits, as well as some sequence of the constituents can be acquired in a single experiment. Indeed, we report here a successful ECD-based FTICR MS top-down approach that affords the composition, stoichiometry, and partial sequence of a 147 kDa noncovalent protein assembly; namely, the yeast alcohol dehydrogenase (ADH) tetramer.

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…However, expectedly, sequence coverage was considerably more comprehensive using CAD and IRMPD in [332] than our approach, involving HCD AIF. In our study, similar to the previous reports [296,332], the sequence origin of the detected fragment ions can be correlated to the outer surface regions of the complex and protein termini.…”

“…The authors correlated this fragmentation pattern with the X-ray crystal structure of the ADH tetramer that showed that the N terminus of the protein is exposed and available for fragmentation, while the C terminus is hidden and involved in the interface of the subunits. Fragmentation coverage was also limited as a result of the formation of charge-reduced ADH1 tetramer species by ECD, resulting in no dissociation of ADH1 subunits from the tetrameric complex [296]. In our study, in addition to N-terminal fragments we also observed C-terminal fragment ions (Figure 4-6A).…”

“…A previous study of ADH1 tetramers by direct infusion native MS on a Fourier transform ion cyclotron resonance (FTICR)-MS with electron capture dissociation (ECD) reported on the fragmentation of residues exclusively at the N-terminus of the protein [296]. The authors correlated this fragmentation pattern with the X-ray crystal structure of the ADH tetramer that showed that the N terminus of the protein is exposed and available for fragmentation, while the C terminus is hidden and involved in the interface of the subunits.…”

“…In native MS, noncovalent interactions are preserved, and tertiary and quaternary structures maintained [74,295]. Recently, native MS has emerged as a powerful tool to provide information on topology, stoichiometry, function, dynamics, and composition of protein assemblies [74,91,296]. In more conventional top-down and intact MS characterization of proteins, non-covalent associations are lost as the protein is unfolded under denaturing conditions.…”

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS

“…In these experiments, charge reduction and the subsequent transmission and detection of the resulting extremely high m/z species typically require instrument modifications [15,18]. Limited charge reduction is also typically observed as a side reaction in electron capture (ECD) and electron transfer dissociation (ETD) experiments, without instrument modification, particularly when working with intact proteins [19][20][21][22][23][24]. In addition, ECD or ETD fragments can remain bound to each other by noncovalent interactions so that they appear as chargereduced precursor ions in the spectra [24][25][26][27].…”