25 research outputs found
Condenser:a platform for handling quantitative proteomic data from matrix science's mascot distiller
Direct identification of functional amyloid proteins by label-free quantitative mass spectrometry
Functional amyloids are important structural and functional components of many biofilms, yet our knowledge of these fascinating polymers is limited to a few examples for which the native amyloids have been isolated in pure form. Isolation of the functional amyloids from other cell components represents a major bottleneck in the search for new functional amyloid systems. Here we present a label-free quantitative mass spectrometry method that allows identification of amyloid proteins directly in cell lysates. The method takes advantage of the extreme structural stability and polymeric nature of functional amyloids and the ability of concentrated formic acid to depolymerize the amyloids. An automated data processing pipeline that provides a short list of amyloid protein candidates was developed based on an amyloid-specific sigmoidal abundance signature in samples treated with increasing concentrations of formic acid. The method was evaluated using the Escherichia coli curli and the Pseudomonas Fap system. It confidently identified the major amyloid subunit for both systems, as well as the minor subunit for the curli system. A few non-amyloid proteins also displayed the sigmoidal abundance signature. However, only one of these contained a sec-dependent signal peptide, which characterizes most of all secreted proteins, including all currently known functional bacterial amyloids
Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild-type rats
Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregationâprone proteins such as αSN, which is central in the development of Parkinson's disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThTâpositive aggregates that contain higherâorder ÎČâsheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9âkDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to crossâseed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes
Influence of p-cresol on the proteome of the autotrophic nitrifying bacterium Nitrosomonas eutropha C91
Identification of Putative Genes Involved in Bisphenol A Degradation Using Differential Protein Abundance Analysis of Sphingobium sp. BiD32
Discharge
of the endocrine disrupting compound bisphenol A (BPA)
with wastewater treatment plant (WWTP) effluents into surface waters
results in deleterious effects on aquatic life. Sphingobium sp. BiD32 was previously isolated from activated sludge based on
its ability to degrade BPA. This study investigated BPA metabolism
by Sphingobium sp. BiD32 using label-free
quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a species-specific platform for
optimal protein identification. The bacterial proteomes of Sphingobium sp. BiD32 in the presence and absence
of BPA were identified and quantified. A total of 2155 proteins were
identified; 1174 of these proteins were quantified, and 184 of these
proteins had a statistically significant change in abundance in response
to the presence/absence of BPA (<i>p</i> †0.05).
Proteins encoded by genes previously identified to be responsible
for protocatechuate degradation were upregulated in the presence of
BPA. The analysis of the metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated metabolite.
A novel <i>p</i>-hydroxybenzoate hydroxylase enzyme detected
by proteomics was implicated in the metabolic pathway associated with
the detected metabolite. This enzyme is hypothesized to be involved
in BPA degradation by Sphingobium sp.
BiD32, and may serve as a future genetic marker for BPA degradation