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Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

ABSTRACT: Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins.

SUBMITTER: Choksawangkarn W

PROVIDER: S-EPMC3586300 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

Choksawangkarn Waeowalee W Kim Sung-Kyoung SK Cannon Joe R JR Edwards Nathan J NJ Lee Sang Bok SB Fenselau Catherine C

Journal of proteome research 20130131 3

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular ...[more]

PMID: 23289353

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Project description:BackgroundRespirable crystalline silica (silicon dioxide; SiO?, quartz) particles are known to induce chronic inflammation and lung disease upon long-term inhalation, whereas non-crystalline (amorphous) SiO? particles in the submicrometre range are regarded as less harmful. Several reports have demonstrated that crystalline, but also non-crystalline silica particles induce IL-1? release from macrophages via the NALP3-inflammasome complex (caspase-1, ASC and NALP3) in the presence of lipopolysaccharide (LPS) from bacteria. Our aim was to study the potential of different non-crystalline SiO? particles from the nano- to submicro-sized range to activate IL-1? responses in LPS-primed RAW264.7 macrophages and primary rat lung macrophages. The role of the NALP3-inflammasome and up-stream mechanisms was further explored in RAW264.7 cells.ResultsIn the present study, we have shown that 6 h exposure to non-crystalline SiO? particles in nano- (SiNPs, 5-20 nm, 50 nm) and submicro-sizes induced strong IL-1? responses in LPS-primed mouse macrophages (RAW264.7) and primary rat lung macrophages. The primary lung macrophages were more sensitive to Si-exposure than the RAW-macrophages, and responded more strongly. In the lung macrophages, crystalline silica (MinUsil 5) induced IL-1? release more potently than the non-crystalline Si50 and Si500, when adjusted to surface area. This difference was much less pronounced versus fumed SiNPs. The caspase-1 inhibitor zYVAD and RNA silencing of the NALP3 receptor reduced the particle-induced IL-1? release in the RAW264.7 macrophages. Furthermore, inhibitors of phagocytosis, endosomal acidification, and cathepsin B activity reduced the IL-1? responses to the different particles to a similar extent.ConclusionsIn conclusion, non-crystalline silica particles in the nano- and submicro-size ranges seemed to induce IL-1? release from LPS-primed RAW264.7 macrophages via similar mechanisms as crystalline silica, involving particle uptake, phagosomal leakage and activation of the NALP3 inflammasome. Notably, rat primary lung macrophages were more sensitive with respect to silica-induced IL-1? release. The differential response patterns obtained suggest that silica-induced IL-1? responses not only depend on the particle surface area, but on factors and/or mechanisms such as particle reactivity or particle uptake. These findings may suggest that bacterial infection via LPS may augment acute inflammatory effects of non-crystalline as well as crystalline silica particles.

Dataset Information

Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

Publications

Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

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