Project description:Global profiling of phosphoproteomes has proven to be a great challenge due to the relatively low stoichiometry of protein phosphorylation and poor ionization efficiency in mass spectrometers. Effective, physiologically relevant, phosphoproteome research relies on the efficient phosphopeptide enrichment from complex samples. Immobilized metal affinity chromatography and titanium dioxide chromatography can greatly assist selective phosphopeptide enrichment. However, the complexity of resultant enriched samples is often still high, suggesting that further separation of enriched phosphopeptides is required. We have developed a pH gradient elution technique for enhanced phosphopeptide identification in conjunction with titanium dioxide chromatography. Using this process, we demonstrated its superiority to the traditional "one-pot" strategies for differential protein identification. Our technique generated a highly specific separation of phosphopeptides by an applied pH gradient between 9.2 and 11.3. The most efficient elution range for high-resolution phosphopeptide separation was between pHs 9.2 and 9.4. High-resolution separation of multiply phosphorylated peptides was primarily achieved using elution ranges greater than pH 9.4. Investigation of phosphopeptide sequences identified in each pH fraction indicated that phosphopeptides with phosphorylated residues proximal to acidic residues, including glutamic acid, aspartic acid, and other phosphorylated residues, were preferentially eluted at higher pH values.
Project description:In order to evaluate the identification of genes and pathways, the global gene expression profiles were assessed in response to UV, TiO2 and UV+TiO2 on nematode, Caenorhabditis elegans. We performed whole genome DNA microarray experiments using age synchronized young adult C. elegans population exposed to UV, TiO2 and UV+TiO2 for 24h. We used whole genome microarrays to screen for global changed in C. elegans transcription profiles and with subsequent quantitative analysis conducted on selected genes.
Project description:Phosphopeptides were enriched from STMN1 knockdown cells via the TiO2 HAMMOC approach, followed by SWATH-MS data acquisition for comparative proteomics analysis.
Project description:In order to evaluate the identification of genes and pathways, the global gene expression profiles were assessed in response to UV, TiO2 and UV+TiO2 on nematode, Caenorhabditis elegans. We performed whole genome DNA microarray experiments using age synchronized young adult C. elegans population exposed to UV, TiO2 and UV+TiO2 for 24h. We used whole genome microarrays to screen for global changed in C. elegans transcription profiles and with subsequent quantitative analysis conducted on selected genes. Young adults C. elegans were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Pilotexp_IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_FT_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The unphosphorylated peptides found in the flowthrough of the TiO2 column were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. The iTRAQ ratio obtained for the unmodified peptides from this flowthrough sample was used to calculate protein ratios for the 4 different conditions.
Project description:The nematode C. elegans was exposed to TiO2 nanoparticles (NPs) to evaluate the ecotoxicity of TiO2 nanoparticles. We used the DNA microarray method to understand changes in gene expression after the exposure to TIO2 NPs. We identified various genes involved in metal detoxification as well as in regulating worm development.
Project description:Changes in phosphorylation of specific sites were examined by TiO2-based phosphopeptide enrichment from wild-type and PKD2/3-deficient thymocytes followed by LC-MS/MS analysis. [Original project description]
Project description:Changes in phosphorylation of specific sites were examined by TiO2-based phosphopeptide enrichment from wild-type and PKD2/3-deficient thymocytes followed by LC-MS/MS analysis. [Original project description]
Project description:Changes in phosphorylation of specific sites were examined by TiO2-based phosphopeptide enrichment from wild-type and PKD2/3-deficient thymocytes followed by LC-MS/MS analysis. [Original project description]
Project description:This work reports highly selective phosphopeptide enrichment using amorphous TiO2 nanotubes (TiO2NTs) and the same material decorated with superparamagnetic Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs). TiO2NTs and TiO2NTs@Fe3O4NPs materials were applied for phosphopeptide enrichment both from a simple peptide mixture (tryptic digest of bovine serum albumin and α-casein) as well as from a complex peptide mixture (tryptic digest of Jurkat T cell lysate). The obtained enrichment efficiency and selectivi-ty for phosphopeptides of TiO2NTs and TiO2NTs@Fe3O4NPs was excellent compared to those of the well-established TiO2 micro-spheres. The enrichment protocol was extended for a second elution step facilitating the identification of additional phosphopep-tides. It further turned out that both types of amorphous TiO2 nanotubes provide qualitatively new physico-chemical features that are clearly advantageous for highly selective phosphopeptide enrichment. This has been confirmed experimentally resulting in substantial reduction of non-phosphorylated peptides in the enriched samples. In addition, TiO2NTs@Fe3O4NPs combine a high selectivity with ease of handling due to the superparamagnetic character of the material. The presented materials and performances are further promising for applications towards a whole range of other types of biomolecules to be treated in similar fashion.