Dataset Information

Direct identification of functional amyloid proteins in cell lysate by label-free quantitative mass spectrometry

ABSTRACT: Functional amyloids are important structural and functional components in many biofilm, yet our knowledge of these fascinating polymers is limited to a few examples for which the amyloid have been purified and characterized by mass spectrometry. Isolation of the functional amyloid from other cell components represents the major bottleneck in the identification of novel functional amyloids. Here we present a label-free quantitative mass spectrometry based method that allows identification of amyloid proteins directly in cell lysates treated with increasing concentrations of formic acid (0-100%). Many functional amyloids are only depolymerized in the presence of concentrated formic acid and provide a characteristic sigmoidal signature when protein abundance is plotted against the formic acid concentration. An automated data processing pipeline was developed to provide a shortlist of amyloid protein candidates based on the amyloid specific abundance signature. The method was evaluated using the E. coli curli and the Pseudomonas Fap system. The major amyloid subunit was confidently identified for both systems and the minor subunit was also identified for the curli system. In addition, very few false positive candidates were identified and these could easily be discarded bases on bioinformatics analysis. Finally, we provide evidence for the application of the method for complex samples with low diversity. This provides an opportunity to identify amyloid in e.g. clinical biofilms.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Pseudomonas Sp. Uk4 Escherichia Coli

SUBMITTER: Florian-Alexander Herbst

LAB HEAD: Per Halkjær Nielsen

PROVIDER: PXD006835 | Pride | 2017-08-09

REPOSITORIES: Pride

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Direct Identification of Functional Amyloid Proteins by Label-Free Quantitative Mass Spectrometry.

Danielsen Heidi N HN Hansen Susan H SH Herbst Florian-Alexander FA Kjeldal Henrik H Stensballe Allan A Nielsen Per H PH Dueholm Morten S MS

Biomolecules 20170804 3

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 c ...[more]

PMID: 28777328

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Project description:Cardiac amyloidosis is a severe clinical condition leading to restrictive cardiomyopathy and heart failure. With increasing options for specific treatment of different cardiac amyloid diseases, correct amyloid subtyping is essential for clinical outcome. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods typically include laser-capture microdissection to ensure the specific analysis of amyloid deposits. While this strategy effectively reduces background signals, it is a labor-intensive and costly procedure hampering the implementation of these methods into a wider group of laboratories. Here we introduce a more direct proteomics-based method for subtyping of cardiac amyloids. Endomyocardial biopsies were retrospectively analyzed from fresh frozen material of 78 patients with cardiac amyloidosis and from 12 biopsies of unused donor heart explants. Cryostat sections were directly digested with trypsin and analyzed with the nano-liquid chromatography (nLC-MS/MS), and the data were evaluated by a proteomic software. With a diagnostic threshold set to 70% for each of the four most common amyloid proteins affecting the heart (LC k, LC l, TTR and SAA), 65 of the cases (87%) could be diagnosed, and of these, 61 cases (94%) were in concordance with the original diagnoses. The specimens were also analyzed for the summed intensities of the amyloid signature proteins (ApoE, ApoA-IV and SAP). The summed intensities were significantly higher (p<0.001) for all assigned amyloid cases, whereas unassigned cases were not significantly different from controls and had lower signals than the assigned cases (p<0.001), suggesting that signature proteins can serve as in situ quality markers of cardiac amyloid deposits. We argue that this efficient workflow can be useful for disseminating proteomics for cardiac amyloid subtyping into accredited clinical laboratories.

			Action	DRS
	EColi_Curli.rar	Other
	Ecoli_SM2258_0_FA1.raw	Raw
	Ecoli_SM2258_0_FA2.raw	Raw
	Ecoli_SM2258_0_FA3.raw	Raw
	Ecoli_SM2258_0_FA4.raw	Raw