Phosphorylation identification of the wild type Wzc protein from E.coli
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia Coli
SUBMITTER: 
Yihua Huang
PROVIDER: PXD080036 | iProX | Tue Jun 23 00:00:00 GMT+01:00 2026
REPOSITORIES: iProX
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Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.
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2004-02-11 | GSE1012 | GEO
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2025-08-21 | PXD054143 | Pride
Project description:For proteomics analysis, testes of Ube2j2-/- and WT mice were collected on ice, washed once with pre-cooled PBS, dried with absorbent paper, and immediately placed in liquid nitrogen, dry ice, or at -80 ℃. Samples were packed in dry ice and transported to Hangzhou Jingjie Biotechnology Company (PTM Biolabs, Inc.) for whole protein 4D label-free quantitative proteomic determination. Testes of Ube2j2-/- and WT mice were subjected to LC-MS/MS analysis following a standard protocol. The sample was grinded and mixed with lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail III (Merck Millipore, 539134) dissolved in aqueous solution containing buffer salts), followed by sonication on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Then, the supernatant was collected and the protein concentration was determined with BCA kit (Beyotime Biotechnology, P0011) according to the manufacturer’s instructions. The sample was slowly added to a final concentration of 20% (m/v) trichloroacetic acid (Sigma-Aldrich, T4885) to precipitate proteins, then mixed by vortexing and incubated for 2 h at 4 °C. The precipitate was collected by centrifugation at 4500 x g for 5 min at 4°C. The precipitated protein was washed 3 times with pre-cooled acetone and dried for 1 min. Protein samples were then redissolved in 200 mM TEAB (Triethylammonium bicarbonate buffer, Sigma-Aldrich, T7408) and ultrasonically dispersed. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight. The sample was reduced with 5 mM dithiothreitol for 60 min at 37 °C and alkylated with 11 mM iodoacetamide for 45 min at room temperature in darkness. Finally, the peptides were desalted using a Strata X SPE column (Phenomenex). The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). The released peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Maxquant 1.6.15.0 software was used for spectra analysis, database searches, and peptide identification.
2025-06-05 | PXD063637 | Pride
Project description:HEK293T cells that transfected with plasmid encoding human FLAG-Granzyme A (Sino Biological, HG13325-NF) using lipofectamine 3000 for 24 h were treated with 4-OI (0.5 mM) for 6h.Following treatment, cells were lysed in lysis buffer and immunoprecipitated with anti-FLAG antibody and protein A/G agarose beads. Next the beads were washed three times with 1ml lysis buffer after centrifuging at 400g for 2 min. The immune complexes were eluted by addition of 40 ml lysis buffer, dissolved in 1x SDS sample buffer and boiled for 5 min. The samples were separate by SDS-PAGE and subsequently probed with Coomassie blue. The corresponding bands for FLAG-Granzyme A were excised from the gel and subjected to in-gel digest with trypsin. Briefly, the gel slices were cut into smaller pieces before reduction with dithiothreitol (10 mM) and then dehydrated in 100% acetonitrile. Gel slices were digested with trypsin overnight at 37℃. Peptides were eluted from the gel pieces following protease digest and dried down completely in a vacuum centrifuge. Samples were analyzed in a Q Exactive TM Plus (Thermo Fisher) coupled online to UPLC after the nanospray ionization source. MS data was analyzed with Proteome Discoverer 1.3 and tandem mass spectra were searched against the UniProt database. Trypsin/P is designated as a cleavage enzyme that allows up to two deletion cleavage. Precursor mass tolerance was set to 10 ppm, while fragments were detected with a tolerance of 0.02 Da.
2023-11-29 | PXD047364 |
Project description:Samples were reduced by the addition of TCEP (25 mM final concentration) and incubated at 37 ºC for 10 min. Alkylation was carried out by the addition of iodoacetamide (25 mM final concentration) and incubated at RT for 1 h in the dark. Samples were then digested by the addition of 1:50 LysC (Wako, Japan) in 100 mM triethylammonium bicarbonate (TEAB) followed by incubation at 37 ºC for 6h and then addition of 1:50 trypsin (Thermo) followed by incubation at 37 ºC overnight. The efficiency of sample digestion efficiency was assessed by MS (LTQ XL, Thermo). The samples were then vacuum-dried and resuspended in 100 mM TEAB (100 µL). Samples were then incubated in their respective Tandem Mass Tag™ (TMT) 10-plex reagents (Thermo) for 1 h at RT with agitation. Reactions were quenched by the addition of 5% hydroxylamine for 15 min and each set of samples (treated and vehicle) were pooled in the same tube and dried overnight. The TMTTM-labelled samples were dried and kept at -85 ºC until further analysis.
2021-07-18 | PXD023780 | Pride