Project description:N-terminomics of mitochondrial proteins using subtiligase
Additional variable modifications:
Abu (L-aminobutyric acid) (+85.05276 Da)
Project description:N-terminomics of mitochondrial proteins using subtiligase
Additional variable modifications:
Abu (L-aminobutyric acid) (+85.05276 Da)
Project description:N-terminomics of mitochondrial proteins using subtiligase
Additional variable modifications:
Abu (L-aminobutyric acid) (+85.05276 Da)
Project description:We surveyed the heterogeneity of the mitochondrial proteome and its function during a typical night and day cycle in Arabidopsis shoots. This used a staged, quantitative analysis of the proteome across 10 time points covering 24 h of the life of 3-week-old Arabidopsis shoots grown under 12-h dark and 12-h light conditions. Results were queried against an in-house Arabidopsis database comprising ATH1.pep (release 7) from The Arabidopsis Information Resource and the Arabidopsis mitochondrial and plastid protein sets (the combined database contained a total of 30,700 protein sequences with 12,656,682 residues) using the Mascot search engine version 2.2 and utilizing error tolerances of +-1.2 Da for MS and +-0.6 Da for MS/MS, 'enzyme' set to trypsin, 'maximum missed cleavages' set to 1, variable modifications of oxidation (Met) and carbamidomethyl (Cys), instrument set to ESI-TRAP, and peptide charge set at 2+ and 3+. ATH1.pep is a non-redundant database with systematically named protein sequences based on Arabidopsis genome sequencing and annotation. PRIDE XML files with accession numbers 10471–10525 and without peptide/protein identifications have already been made public.
Project description:S.cerevisiae proteomes were prepared for LC-MS/MS analysis using an Ultimate 3000 HPLC system (Dionex, Amsterdam, The Netherlands) in-line connected to a LTQ Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany). Mascot server version 2.2 from Matrix Science was then used to identify the MS/MS spectra in the S. cerevisiae content of UniProtKB/Swiss-Prot (version 15.10) concatenated with the 5-UTR peptide centric database derived from SGD. A shuffled version of this concatenated database was created to estimate the false discovery rate in the results at 0.64%. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.5 Da. Semi-specific Arg-C/P was used as enzyme specificity and no missed cleavages were allowed. The fixed modifications were 13C2D3-acetylation (+47 Da) on Lys, carbamidomethylation (+57 Da) on Cys and oxidation (+16 Da) on Met, and the variable modifications were acetylation (+42 Da) and 13C2D3-acetylation (+47 Da) on the N-terminus. N-terminal propionylation (+56 Da) was set as an additional variable modification in a parallel search for N-terminal propionylation. The charge state was set to allow single, double and triple charged peptides. All peptide identifications were subsequently processed, stored and managed by ms-lims.
Project description:Comparison of the extraction performances of three different mitochondria enrichment protocols (differential centrifugation, sucrose gradient, a commercial kit based on surfactants) on ten different cell lines. Samples were analysed on several instrumental platforms.
Mitochondrial pellets were lysed in RapiGest 0.1% (RG, Waters Corporation) and digested with trypsin in 50mM ammonium bicarbonate. Before digestion the proteins were first quantified according to the Bradford assay and then reduced with 1mM TCEP 50 mM and alkylated wit IAA 20mM. Peptides were recovered by centrifugation and then loaded directly on the respective chromatographic system for the mass spectrometry analysis or on a SDS-PAGE for WB analysis.
Raw data were processed with PEAKS 7.5 with the following search parameters: Parent Mass Error Tolerance: 10.0 ppm; Fragment Mass Error Tolerance: 0.05 Da; Enzyme specificity: Trypsin; Max Missed Cleavages: 2; Non-specific Cleavage: one; Fixed Modifications: Carbamidomethylation (C); Variable Modifications: Oxidation (M), Acetylation (K) Ubiquitin; Deamidation (NQ); Max variable PTM per peptide: 2. The database searched was custom made and contained Uniprot Swiss Prot Human, common contaminants and yeast enolase (20442 entries).
Project description:Comparison of the extraction performances of three different mitochondria enrichment protocols (differential centrifugation, sucrose gradient, a commercial kit based on surfactants) on ten different cell lines. Samples were analysed on several instrumental platforms.
Mitochondrial pellets were lysed in RapiGest 0.1% (RG, Waters Corporation) and digested with trypsin in 50 mM ammonium bicarbonate. Before digestion the proteins were first quantified according to the Bradford assay and then reduced with 1 mM TCEP 50 mM and alkylated wit IAA 20 mM. Peptides were recovered by centrifugation and then loaded directly on the respective chromatographic system for the mass spectrometry analysis or on a SDS-PAGE for WB analysis.
Raw data were processed with PEAKS 7.5 with the following search parameters.
Parent Mass Error Tolerance - depending on the instrument - 10.0 to 40 ppm
Fragment Mass Error Tolerance - depending on the instrument - 0.05 to 0.6 Da
Enzyme specificity Trypsin
Max Missed Cleavages: 2
Non-specific Cleavage: 1
Fixed Modifications: Carbamidomethylation (C)
Variable Modifications: Oxidation (M), Deamidation (NQ)
Max variable PTM per peptide: 2
The searched database was downloaded from www.nextprot.org and contained the latest annotated human proteome including isoforms (42,151 entries).
Project description:<p> Human disorders of mitochondrial oxidative phosphorylation (OXPHOS) represent a devastating collection of inherited diseases. These disorders impact at least 1:5000 live births, and are characterized by multi-organ system involvement. They are characterized by remarkable locus heterogeneity, with mutations in the mtDNA as well as in over 77 nuclear genes identified to date. It is estimated that additional genes may be mutated in these disorders. </p> <p>To discover the genetic causes of mitochondrial OXPHOS diseases, we performed targeted, deep sequencing of the entire mitochondrial genome (mtDNA) and the coding exons of over 1000 nuclear genes encoding the mitochondrial proteome. We applied this 'MitoExome' sequencing to 124 unrelated patients with a wide range of OXPHOS disease presentations from the Massachusetts General Hospital Mitochondrial Disorders Clinic. </p> <p>The 2.3Mb targeted region was captured by hybrid selection and Illumina sequenced with paired 76bp reads. The total set of 1605 targeted nuclear genes included 1013 genes with strong evidence of mitochondrial localization from the MitoCarta database, 377 genes with weaker evidence of mitochondrial localization from the MitoP2 database and other sources, and 215 genes known to cause other inborn errors of metabolism. Approximately 88% of targeted bases were well-covered (>20X), with mean 200X coverage per targeted base. </p>
Project description:Mitochondria are organelles that generate most of the energy in eukaryotic cells in the form of ATP via oxidative phosphorylation in eukaryote. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA are required to translate essential subunits of the respiratory chain complexes involved in oxidative phosphorylation. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. These modifications are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. We performed a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species, and revised the modification status of some of the previously studied species. In total, we found 17 kinds of RNA modifications at 137 positions (8.7% in 1,575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for human mt-tRNA modifications are provided. We here demonstrated that both QTRT1 and QTRT2 are required for biogenesis of queuosine (Q) at position 34 of four mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the mitochondrial decoding system, and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.