Quantitative TMT phosphoproteomics in neonatal mouse ventricular myocytes (NMVMs) upon NUAK1 overexpression
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Yi-Gang Li
PROVIDER: PXD074878 | iProX | Wed Feb 25 00:00:00 GMT 2026
REPOSITORIES: iProX
Similar Datasets
Project description:To examined the effect of the antibiotic treatment on protein expression of symbionts in the whitefly. Proteome analysis showed that 23 Hamiltonella proteins were down-regulated in Hamiltonella-cured (–HBt) compared to Hamiltonella-infected (+HBt) whiteflies (Dataset S1A,B), this data including all protein data of Bemisia tabaci and its symbionts. The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). The tandem mass spectra were searched against genome database of Portiera, Hamiltonella and Rickettsia in B. tabaci MEAM1 (http://www.whiteflygenomics.org/cgi-bin/bta/index.cgi) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and oxidation on Met was specified as variable modifications. FDR was adjusted to < 1% and minimum score for peptides was set > 40. KEGG database was used to annotate protein pathway using KEGG online service tools KAAS. Then each protein was mapped to the KEGG pathway database using KEGG online service tools KEGG mapper.
2023-02-14 | PXD038323 | Pride
Project description:The resulting MS/MS data were processed using Proteome Discoverer 1.4 (ThermoFisher). Tandem mass spectra were searched against human caspase-1 protein sequence. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. Mass error was set to 20 ppm for precursor ions and 0.05 Da for fragments ions. Carbamidomethyl on Cys were specified as fixed modification, oxidation on Met and phosphorylation on Ser and Thr were specified as variable modification. Peptide confidence was set at high, and peptide ion score was set >20. The mass spectrometry analysis was performed by Jingjie PTM Biolabs (Hangzhou, China).
2026-04-01 | PXD076488 |
Project description:The resulting MS/MS data were processed using Proteome Discoverer 1.4 (ThermoFisher). Tandem mass spectra were searched against human caspase-1 protein sequence. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. Mass error was set to 20 ppm for precursor ions and 0.05 Da for fragments ions. Carbamidomethyl on Cys were specified as fixed modification, oxidation on Met and phosphorylation on Ser and Thr were specified as variable modification. Peptide confidence was set at high, and peptide ion score was set >20. The mass spectrometry analysis was performed by Jingjie PTM Biolabs (Hangzhou, China).
2026-03-25 | PXD076141 |
Project description:In this study, 3 biological replicates with 3 technical replicates for the conditioned media (CM) and the whole cell lysates (WCL) of C8-D1A cell line were analyzed using 108 LC-MS/MS runs. Each peptide sample was separated into 6 fractions using stageTip based High-pH fractionation. The peptide samples were analyzed using LC-MS/MS instrumentation consisting of a Nanoflow Easy-nLC 1000 that was connected to a Q Exactive mass spectrometer through a nanoelectrospray ion source. All raw files were processed in MaxQuant, version 1.3.0.5 and the Andromeda search engine against the IPI mouse database (version 3.87, 59 534 entries), containing both forward and reverse proteins sequences, and common contaminants. MS/MS searches for the secretome and the whole-cell proteome were performed with the following parameters: carbamidomethylation as a fixed modification; oxidation of methionine and protein N-terminal acetylation as variable modifications; a 20-ppm first-search tolerance; a 6-ppm main-search tolerance. Minimum peptide length was set to six residues. The false discovery rate for all peptides, PTM sites, and protein identifications was set to 0.01.
2014-06-05 | PXD000501 | Pride
Project description:For protein identification, the following parameters were used. Peptide mass tolerance = 20 ppm, Missed cleavage = 2, MS/MS tolerance = 0.1 Da, Enzyme = Trypsin, Fixed modification: Carbamidomethyl (C), iTRAQ8plex(K), iTRAQ8plex(N-term), Variable modification：Oxidation(M)，Decoy database pattern=Reverse. The MASCOT search results for each SCX elution were further processed using the ProteomicsTools (version 3.1.6) which includes the programs BuildSummary, Isobaric Labeling Multiple File Distiller and Identified Protein iTRAQ Statistic Builder (Information can be accessed from Research Center for Proteome Analysis (http://www.proteomics.ac.cn/).
2020-01-27 | PXD000527 | Pride
Project description:To build a reference proteome, we established a BV-2 microglial proteome to a depth of 5494 unique protein groups using a novel strategy that combined FASP, StageTip-based high pH fractionation, and high-resolution mass spectrometry quickly and cost-efficiently. By bioinformatics analysis, the BV-2 proteome is a valuable resource for studies of microglial function, such as in the immune response, inflammatory response, and phagocytosis. Raw files were processed in MaxQuant version 1.2.2.5 and the Andromeda search engine against the IPI mouse database (version 3.87, 59,534 entries) containing both forward and reverse proteins sequences. Carbamidomethylation of cysteines was set as the fixed modification. Oxidation of methionine and acetylation of protein N-term was employed as a variable modification. The first search tolerance was set to 20 ppm, followed by a main search tolerance of 6 ppm. HCD fragment ion mass tolerance was set to 20 ppm. Peptides with a minimum of 6 amino acids were considered for identification. The false discovery rate (FDR) for all peptides, modification sites, and protein identifications were set to 0.01. Gene ontology of identified proteins at FDR < 1% was annotated using the DAVID bioinformatics resource tool and UniprotKB database. Pathway analysis was performed using the KEGG pathway database and Panther pathway database.
2013-09-11 | PXD000168 | Pride
Project description:Plasma proteins were digested with trypsin and the resulting peptides were analyzed by nanoLC–MS/MS on an EASY-nLC 1200 system coupled to an Orbitrap Exploris 480 mass spectrometer. Peptides were loaded onto a self-packed C18 reversed-phase column (25 cm × 100 μm i.d.) and separated at 500 nL/min using a linear gradient with solvent A (0.1% formic acid, 2% acetonitrile in water) and solvent B (0.1% formic acid, 90% acetonitrile in water): 4–20% B (0–68 min), 20–32% B (68–82 min), 32–80% B (82–86 min), and 80% B wash (86–90 min). The MS was operated with FAIMS using compensation voltages of −70 V and −45 V. Full MS scans were acquired at 60,000 resolution over m/z 400–1,200, and MS/MS scans at 30,000 resolution (first mass m/z 110). The top 15 precursors were selected for HCD (NCE 27%) with 30 s dynamic exclusion; AGC target was 75%, intensity threshold 10,000 ions/s, and maximum injection time 100 ms. TurboTMT was disabled for label-free quantification. Raw data were processed in Proteome Discoverer v2.4.1.15 and searched against the Homo_sapiens_9606_PR_20201214.fasta database (75,777 entries) with a reverse decoy strategy. Trypsin was specified with up to two missed cleavages; minimum peptide length was 6 aa. Carbamidomethyl (C) was set as a fixed modification; oxidation (M), protein N-terminal acetylation, methionine loss, and methionine loss + acetylation were set as variable modifications (≤3 variable modifications per peptide). Precursor and fragment mass tolerances were 10 ppm and 0.02 Da, respectively. FDR was controlled at <1% at the PSM, peptide, and protein levels. For label-free quantification, peptide intensities were normalized by peptide-wise mean centering across samples followed by within-sample median normalization, and protein abundances were summarized as the median of normalized peptide values per protein for downstream analyses.
2026-06-21 | PXD073684 | Pride
Project description:We identified the telomere associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitaion (enChIP). Purified proteins were analyzed by GeLC-MS/MS. Tandem mass spectra were extracted by Proteome Discoverer 1.2. Database searchs were submitted to an in-house Mascot server version 2.4.1. Mascot was set up to search the Swissprot database assuming the digestion enzyme as trypsin. Carbamidomethylation of cysteine was set as a fixed modification. Oxidized methionine, acetylation of N terminus and conversion of glutamine to Pyro-Glu at N terminus were set as variable modifications. The precursor mass tolerances were 10 ppm and the tolerance of the MS/MS ions was 0.8 Da. In Scaffold 3.4.5, database search results were grouped according to gel bands.
2013-11-12 | PXD000461 | Pride
Project description:Two datasets: StREM1.3: methyl-beta cyclodextrin-dependent distribution of StREM1.3 to detergent resistant and detergent soluble membrane fractions. SLAH3: localization of SLAH3 to detergent resistant and detergent soluble fractions dependent on co-expression with CPK21 and ABI1. Data analysis: Protein identification and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a fixed modification; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modifications. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identified by MaxQuant were excluded from further analys Protein identification and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a fixed modification; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modifications. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identified by MaxQuant were excluded from further analysis. PXD000211 is also assoicated with the same study/paper.
2013-09-02 | PXD000182 | Pride
Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.
2016-04-04 | PXD002344 | Pride