Dataset Information

Benchmarking of different methods for obtaining high quality secretomics data

ABSTRACT: The main drawback of secretome analyses are contaminating proteins derived from culture medium or proteins released from dying cells which are hard to distinguish from proteins actively secreted by cells. Therefore, several approaches have been applied allowing to remove contamination from secretome analysis. Here, we will compare these approaches and will evaluate their potential to determine proteins secreted by unconventional secretory pathways.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung, Fibroblast Of Dermis, Cell Culture, Mesenchymal Cell

DISEASE(S): Lung Cancer

SUBMITTER: Gereon Poschmann

LAB HEAD: Gereon Poschmann

PROVIDER: PXD018895 | Pride | 2020-10-19

REPOSITORIES: Pride

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Publications

Comparative Secretomics Gives Access to High Confident Secretome Data: Evaluation of Different Methods for the Determination of Bona Fide Secreted Proteins.

Poschmann Gereon G Brenig Katrin K Lenz Thomas T Stühler Kai K

Proteomics 20201117 2

Secretome analysis is broadly applied to understand the interplay between cells and their microenvironment. In particular, the unbiased analysis by mass spectrometry-based proteomics of conditioned medium has been successfully applied. In this context, several approaches have been developed allowing to distinguish proteins actively secreted by cells from proteins derived from culture medium or proteins released from dying cells. Here, three different methods comparing conditioned medium and lysa ...[more]

PMID: 33015975

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Project description:Membrane protein and phospholipid (PL) compositions change in response to environmental cues and during infections. Covalent modification (e.g., unsaturation and cyclopropanation) and remodeling the acyl chain length of PLs is an important bacterial adaptation mechanism. However, little is known about which bacterial pathways are regulated in response to the alteration of PL composition. Here, we show that P. aeruginosa PlaF, which modulates membrane PL composition, is important for biofilm biogenesis, and we performed whole-cell quantitative proteomics of P. aeruginosa wild-type and ∆plaF biofilms to identify pathways regulated by PlaF. The results revealed profound alterations in the abundance of 14 two-component systems (TCS), including the PprA-PprB, which controls the transition to biofilm. Activation of PprA-the PprB system observed by proteomics was confirmed by mRNA quantification. Furthermore, we have observed a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, and differential production of nine proteases in ∆plaF, indicating that PlaF-mediated virulence adaptation involves complex transcriptional and post-transcriptional regulation. Moreover, proteomics revealed that the pyoverdin-mediated iron uptake pathway proteins were depleted in ∆plaF, which agrees with decreased concentrations of extracellular pyoverdine and intracellular iron in ∆plaF and is likely responsible for its prolonged lag phase due to reduced iron uptake. Conversely, the accumulation of proteins from alternative iron-uptake systems in ∆plaF suggests that PlaF may function as a switch between different iron-acquisition pathways. The observation that ∆plaF accumulates PL-acyl chain modifying enzymes and PlsX, an initiator of de novo PL synthesis, reveals novel insights into the relationship between degradation, covalent modification and biosynthesis of PLs for membrane homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that PlaF-catalyzed degradation of PLs is a signal which is amplified by proteins of TCS, phosphorylation and proteolytic degradation systems to elicit the global adaptive response in P. aeruginosa.

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