Label-free quantitative proteomics analysis of ASPC-1 and H358
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Xiufeng Pang
PROVIDER: PXD047389 | iProX | Thu Nov 30 00:00:00 GMT 2023
REPOSITORIES: iProX
Similar Datasets
Project description:Searching parameter: 10 ppm precursor mass tolerance; 0.025 Da product ion mass tolerance; fully digested with trypsin; up to two missed cleavages;
Variable modifications: oxidation of methionine (M+15.9949); heavy lysine (K+8.0142); heavy arginine (R+6.0201);
Fixed modifications: carbamidomethylation of cysteine (C+57.0214); TMT labeling (K+229.1630, N-term+229.1630)
2020-10-20 | MSV000086337 | MassIVE
Project description:Step 1: Cell lysis of yeast sphaeroblast (Saccharomyces cerevisiae) and carbamidomethylation. Step 2: Isotopic labeling with dimethylation (light + medium). Step 3: Proteolytic digestion with trypsin (ArgC specificity because of blocked lysines by dimethylation). Step 4: Enrichment via ChaFRADIC method Step 5: Nano-LC-MS/MS measurements Step 6: Data interpretation. Specifications: --ESI spray ionisation o Q Exactive mass spec. --Quadrupole selection, HCD fragmentation and orbitrap detection o Identification + Quantification: PD version 1.3.0039. Search engine: Mascot 2.4. SGD database (Sep 2011), 6717 target sequences. Special nodes: precursor ions quantifier (within a 1 min retention time window), percolator (default settings). Protease: SemiArgC with max. 2 missed cleavage sites. Used modifications: Yeast_N-term_Enriched_Search_Acetylation Variable -- N-terminal Acetylation (42.0105 Da) -- Dimethylation light (28.0313 Da) at Lys -- Dimethylation medium (34.0689 Da) at Lys fixed -- Carbamidomethylation at Cys (57.0214 Da) Quantification -- Peptides with labeled lysine Yeast_N-term_Enriched_Search_Dimethylation Variable -- N-terminal Dimethylation light (28.0313 Da) -- N-terminal Dimethylation medium (34.0689 Da) -- Dimethylation light (28.0313 Da) at Lys -- Dimethylation medium (34.0689 Da) at Lys fixed -- Carbamidomethylation at Cys (57.0214 Da) Quantification -- Peptides with labeled N-terminus -- Peptides with labeled N-terminus and labeled lysine. Mass tolerance: 10 ppm for MS, 0.02 Da for MS/MS. Applied filters: high confidence (FDR <1%), search engine rank 1
2013-08-09 | PXD000292 | Pride
Project description:Propargyl labeled ubiquitin was immobilized on beads and used for click-chemistry based pull downs. Covalently linked proteins were washed and digested off-bead, or eluted with strong acid and loaded into a SDS-PAGE gel. Digestion with Lys-C, trypsin consequetively and LC-MS/MS. Both dimethyl labeling, as well as qualitative measurements were done. For both the qualitative and the quantitative dataset peak lists were generated from the raw data files using the Proteome Discoverer software package version 1.3.339. Peptide identification was performed by searching the combined peak lists of the entire gel lane (10 bands) against a concatenated target-decoy database containing the human sequences in the Uniprot database (release 2012_06) supplemented with a common contaminants database using the Mascot search engine version 2.3 via the Proteome Discoverer interface. The search parameters included the use of trypsin as proteolytic enzyme allowing up to a maximum of 2 missed cleavages. For the qualitative dataset, carbamidomethylation of cysteines was set as a fixed modification whereas oxidation of methionines was set as variable modification. Precursor mass tolerance was initially set at 50 ppm, while fragment mass tolerance was set at 0.6 Da. Subsequently, the peptide identifications were filtered for true mass accuracy <10 ppm and an ion score of 25. For the quantitative dataset carbamidomethylation of cysteines was set as a fixed modification whereas oxidation of methionines and the dimethyl light and intermediate labels on N-termini and lysine residues were set as variable modifications. Precursor mass tolerance was initially set at 50 ppm, while fragment mass tolerance was set at 0.6 Da for ETD-IT fragmentation and 0.05 Da for HCD and ETD-FT fragmentation. Subsequently, the peptide identifications were filtered for true mass accuracy <10 ppm and an ion score of 25.
2013-08-14 | PXD000073 | Pride
Project description:Carbon metabolism in Mycobacterium smegmatis was analysed using reverse-phase chromatography coupled to orbitrap high-resolution mass spectrometer using trypsin digestion. MS/MS files were searched against the Mycobacterium smegmatis strain MC2-155 database Release 1 - July 2010 ) by MaxQuant software (version 1.3.0.5). Mass spectra were searched with an initial mass tolerance of 7 ppm in MS mode and 0.5 Da in MS/MS mode. Up to two missed cleavages were allowed. Carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (Protein N-term) and Phospho (S, T, Y) were considered as variable modifications. Minimum required peptide length was set to seven amino acids and at least two (unique + razor) peptides were required for protein identification. A cut-off was fixed at 1% FDR at the peptide and protein level. Reverse and contaminants sequences were removed and proteins with a Posterior Error Probability (PEP) lower than 0.1 were accepted for further data treatment. Protein quantification was performed with razor and unique peptides, using only unmodified, oxidated (M) and acetylated (Protein N-term) peptides. A minimum of two ratio counts was required to quantify proteins.
2014-07-09 | PXD000253 | Pride
Project description:Shotgun phosphoproteome of Sulfolobus acidocaldarius using PAciFIC technique. Briefly, proteins were denatured, alkylated, trypsine digestion, SCX separation Fractionated peptides were analysed on Bruker HCTultra. Data processing and bioinformatics: data from mass spectrometry were then extracted to mgf format using Bruker Data Analysis V4.0 with a MRM script, these were then were searched against the Sulfolobus acidocaldarius database (containing 2223 proteins) downloaded from NCBI in March 2010 using Phenyx V 2.6 (Genebio, Geneva). The searches were performed using parameters as follows: carbamidomethylation of cysteine (fixed modification), oxidation of methionine (variation), and phosphorylation of serine, tyrosine, threonine (variation), trypsin with 2 missed cleavages. Furthermore, other parameters such as parent, MS/MS, tolerances were set at 2.0 and 0.8 Da, respectively, whilst minimum peptide length, z-score, p-value and AC score were set at 5, 5.5, 10-5, and 5.5 respectively.
2014-04-15 | PXD000289 | Pride
Project description:The MS raw files were processed and search in Mascot and SEQUEST through the Proteome Discoverer 2.1 software (Thermo Fisher Scientific). Database searches were performed using the following parameters: Precursor mass tolerance of 10 parts per million (ppm); MSMS mass tolerance of 0.05 Da; Enzyme: Trypsin and up to two missed cleavages were allowed.
2019-03-01 | MSV000083506 | MassIVE
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).
2017-05-30 | MSV000081121 | MassIVE
Project description:In order to explore the effects of Rabggta or Ggps1 on the membrane proteome of T cells, sorted CD4+ T cells of SRBCs-immunized Rabggta-icKO, Ggps1-icKO and their WT mice were subjected to membrane protein extraction with standard protocol. Take 50 μg protein and diluted to 100 μL water. Then add 5 μL (0.25 μg/μL) trpsin enzyme and incubate in a 37℃ constant temperature incubator for 16 hours for protein digestion. Then samples were subjected to Nano LC-MS/MS analysis. The raw MS files were analyzed and searched against target protein database based on the species of the samples using MaxQuant. The parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable), Acetyl (N-term) (variable); the enzyme specificity was set to trypsin; the maximum missed cleavages were set to 2; the precursor ion mass tolerance was set to 20 ppm, and MS/MS tolerance was 20 ppm. Only high confident identified peptides were chosen for downstream protein identification analysis. Gene ontology (GO) terms were used to obtain and classify the function information of the DEPs.
2025-04-07 | PXD062705 |
Project description:We investigated the differential phosphoproteome of the mouse heart after isoproterenol stimulus of the AC3-I and AC3-C mice. The former is a model of specific in vivo CaMKII inhibition by a transgenically expressed peptide, whereas the latter is a transgenic mouse expressing a control peptide. Data processing: all raw data files of the individual SCX fractions of each of the 2 mouse experiments were imported into Proteome Discoverer v1.3.0.339 and the combined peak list was split into CID and HCD data (where applicable) before database searching. Subsequently, CID and HCD peak lists were searched individually against an International Protein Index (IPI; http://www.ebi.ac.uk/ipi) database containing mouse sequences and common contaminants such as bovine serum albumin and human keratins (IPI-Mouse v3.84; 60 248 sequences) through a direct connection to our in-house Mascot server (Mascot v2.3.2, Matrix Science, London, UK). The following settings were used: carbamidomethylation on cysteines as static modification; light, intermediate, and heavy dimethylation of peptide N-termini and lysine side chains, as well as oxidation on methionine and phosphorylation on serine, threonine, or tyrosine as variable modifications; and precursor mass tolerance of 20 ppm and 0.8 Da on the fragment masses (for CID) but 20 ppm and 0.02 Da for HCD searching. The enzyme was specified as trypsin, and 2 missed cleavages were allowed.
2013-08-02 | PXD000174 | Pride
Project description:All samples derived from the q-TIP experiments were analysed using reverse-phase chromatography coupled to orbitrap high-resolution mass spectrometer using trypsin digestion. MS/MS files were searched against the UniProt human protein database (release date 18-04-2012) by MaxQuant software (version 1.2.2.5 and 1.3.0.5). Mass spectra were searched with an initial mass tolerance of 7 ppm in MS mode and 0.5 Da in MS/MS mode. Up to two missed cleavages were allowed. Carbamidomethylation was set as a fixed modification, whereas oxidation (M) and acetylation (Protein N-term) were considered as variable modifications. Phospho (STY) was set as variable modifications for the approach B as well. Minimum required peptide length was set to six amino acids (seven for the approach B) and at least two (unique + razor) peptides were required for protein identification. A cut-off was fixed at 1% FDR at the peptide and protein level. Reverse and contaminants sequences were removed and proteins with a Posterior Error Probability (PEP) lower than 0.1 were accepted for further data treatment. Protein quantification was performed with razor and unique peptides, using only unmodified, oxidated (M) and acetylated (Protein N-term) peptides. A minimum of two ratio counts was required to quantify proteins. A FDR < 0.05 was applied on the Significances B to accept proteins significantly differentially quantified between conditions. In the article and the supplementary table 2, Rep3 corresponds to the forward experiment or the second replicate (Rep2)
2013-11-26 | PXD000243 | Pride