Article
A triaxial probe for on-line proteolysis coupled with hydrogen/deuterium exchange-electrospray mass spectrometry.
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, USA.
Journal of the American Society for Mass Spectrometry (impact factor:
4).
03/2007;
18(2):208-17.
DOI:10.1016/j.jasms.2006.09.011
pp.208-17
Source: PubMed
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Citations (0)
- Cited In (2)
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Article: Biophysical analyses of synthetic amyloid-beta(1-42) aggregates before and after covalent cross-linking. Implications for deducing the structure of endogenous amyloid-beta oligomers.
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ABSTRACT: A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that soluble Abeta aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Abeta aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Abeta oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Abeta have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Abeta aggregates in vitro that resemble the endogenous soluble Abeta aggregates, we previously found that monomeric Abeta(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Abeta(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Abeta cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Abeta(1-42) with three cross-linking agents: transglutaminase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS.Biochemistry 11/2009; 48(49):11796-806. · 3.42 Impact Factor -
Article: Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.
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ABSTRACT: Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.Molecular & Cellular Proteomics 02/2011; 10(2):M110.001479. · 7.40 Impact Factor
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Keywords
aid localization
coaxial probe
HDX-MS studies
hydrogen/deuterium exchange-mass spectrometry
hydrolysis performance
on-line proteolysis system utilizing
organic solvent component
protein aggregates
triaxial electrospray probe
