ovalbumin and ferrous-oxide

Nanoparticle (NP)-based vaccines are attractive immunotherapy tools because of their capability to codeliver antigen and adjuvant to antigen-presenting cells. Their cellular distribution and serum protein interaction ("protein corona") after systemic administration and their effect on the functional properties of NPs is poorly understood.. We analyzed the relevance of the protein corona on cell type-selective uptake of dextran-coated NPs and determined the outcome of vaccination with NPs that codeliver antigen and adjuvant in disease models of allergy.. The role of protein corona constituents for cellular binding/uptake of dextran-coated ferrous nanoparticles (DEX-NPs) was analyzed both in vitro and in vivo. DEX-NPs conjugated with the model antigen ovalbumin (OVA) and immunostimulatory CpG-rich oligodeoxynucleotides were administered to monitor the induction of cellular and humoral immune responses. Therapeutic effects of this DEX-NP vaccine in mouse models of OVA-induced anaphylaxis and allergic asthma were assessed.. DEX-NPs triggered lectin-induced complement activation, yielding deposition of activated complement factor 3 on the DEX-NP surface. In the spleen DEX-NPs targeted predominantly B cells through complement receptors 1 and 2. The DEX-NP vaccine elicited much stronger OVA-specific IgG. Opsonization of lectin-coated NPs by activated complement components results in selective B-cell targeting. The intrinsic B-cell targeting property of lectin-coated NPs can be exploited for treatment of allergic immune responses.

Topics: Anaphylaxis; Animals; Antigens; B-Lymphocytes; Dextrans; Drug Carriers; Female; Ferrous Compounds; Hypersensitivity; Lectins; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; Protein Corona; T-Lymphocytes; Vaccines

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, conventional IMAC lacks enough specificity for efficient phosphoproteome analysis. In this study, novel Fe 3O 4@TiO 2 microspheres with well-defined core-shell structure were prepared and developed for highly specific purification of phosphopeptides from complex peptide mixtures. The enrichment conditions were optimized using tryptic digests of beta-casein, and the high specificity of the Fe 3O 4@TiO 2 core-shell microspheres was demonstrated by effectively enriching phosphopeptides from the digest mixture of alpha-casein and beta-casein, as well as a five-protein mixture containing nonphosphoproteins (bovine serum albumin (BSA), myoglobin, cytochrome c) and phosphoproteins (ovalbumin and beta-casein). The Fe 3O 4@TiO 2 core-shell microspheres were further successfully applied for the nano-LC-MS/MS analysis of rat liver phosphoproteome, which resulted in identification of 56 phosphopeptides (65 phosphorylation sites) in mouse liver lysate in a single run, indicating the excellent performance of the Fe 3O 4@TiO 2 core-shell microspheres.

Topics: Amino Acid Sequence; Animals; Caseins; Cytochromes c; Ferric Compounds; Ferrous Compounds; Hydrogen-Ion Concentration; Iron Compounds; Liver; Microspheres; Molecular Sequence Data; Myoglobin; Ovalbumin; Phosphopeptides; Phosphoproteins; Phosphorylation; Proteomics; Rats; Serum Albumin, Bovine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Titanium; Trypsin

The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe(3)O(4)/SiO(2) core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol beta-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe(3)O(4) nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe(3)O(4) nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of alpha-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.

Topics: Animals; Caseins; Data Interpretation, Statistical; Databases, Protein; Ferrous Compounds; Glass; Hydrolysis; Indicators and Reagents; Liver; Magnetics; Mice; Nanoparticles; Organophosphonates; Ovalbumin; Phosphoproteins; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Zirconium