Proteomic analysis of lung cancer types
Ontology highlight
ABSTRACT:
INSTRUMENT(S): maXis II
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Lilla Turiak
PROVIDER: MSV000089291 | MassIVE | Thu Apr 21 06:48:00 BST 2022
REPOSITORIES: MassIVE
Similar Datasets
Project description:Samples were analyzed using a Maxis II QTOF instrument (Bruker Daltonik GmbH, Bremen, Germany) equipped with CaptiveSpray nanoBooster ion source coupled to a Dionex UltiMate 3000 RSLCnano system (Sunnyvale, CA, USA). Peptides were separated on an Acquity M-Class BEH130 C18 analytical column using gradient elution (isocratic hold at 4% for 11 min, then elevating B solvent content to 25% in 75 min, and to 40% in 15 min) following trapping on an Acclaim PepMap100C18 trap column. Solvent A consisted of water + 0.1% formic acid, Solvent B was acetonitrile + 0.1% formic acid, and the sample loading buffer was 0.1% TFA and 0.01% HFBA in water.
The MS/MS measurements were carried out in DDA mode. The cycle time was set at 2.5 sec, with a dynamic MS/MS exclusion of the same precursor ion for 2 minutes, or if the intensity is at least 3 times larger. Preferred charge states were set between +2 and +5. MS spectra were acquired at 3 Hz in the 150-2200 m/z range, while MS/MS spectra at 4 or 16 Hz depending on the intensity of the precursor. For single stage MS measurements spectra were recorded over the mass range of 300-3000 m/z at 1 Hz.
2021-04-28 | MSV000087329 | MassIVE
Project description:Leaves of Brachypodium distachyon Bd21 were collected at three leaf stage. The total proteins were extracted and then phosphopeptides were enriched by using TiO2 microcolumns and phosphopeptides and their corresponding proteins were identified with LC-MS/MS and Maxquant software. In details, enriched phosphopeptides were separated on a self-packed C18 reverse phase column (75 μm I.D., 150 mm length) (Column Technology Inc., Fremont, CA), which directly connected the nano electrospray ion source to a LTQ-Orbitrap XL mass spectrometer. Pump flow was split to achieve a flow rate at 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% FA (A) and 0.1% FA and 80% ACN (B). A five-step linear gradient of 5% to 30% B in 105 min, 35% to 90% B in 16 min, 90% B in 4 min, 90% to 2% B for 0.5 min and 2% B for 14.5 min was performed. The spray voltage was set to 2.0 kV and the temperature of the heated capillary was 240ºC. For data acquisition, a data-dependent top 10 MS/MS spectraper MS full scan in the Orbitrapanalyser was used. The raw files were processed with Maxquant (version 1.1.1.36) and searched against the NCBI Brachypodium protein database (26,035 entries in total) concatenated with a decoy of reversed sequences. The following parameters were used for database searches: cysteine carbamidomethylation was selected as a fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/MS spectra.
2013-12-17 | PXD000340 | Pride
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
Project description:Abstract still has to be written. The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 um internal diameter (I.D.) x 20 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 um I.D. x 150 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A' (0.05% formic acid) to 55% solvent B' (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B'. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the human (H. sapiens) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2).
2016-12-23 | PXD000551 | Pride
Project description:Four bile samples collected from patients with a malignant (PAC, CC) or a nonmalignant (CP, BS) biliary stenosis were used for comparative proteomic analysis. Briefly, bile samples were centrifuged at 16,000/g/ for 10 min at 4 C. Each supernatant was delipided with Cleanascite (Biotech Support Group, North Brunswick, NJ, USA) and ultrafiltrated using a 3 kDa filter cut-off (YM-3 centricon, Millipore, Bedford, MA, USA). For each sample, 50 ug of proteins were mixed with 0.5 M triethylammonium bicarbonate (TEAB) buffer pH 8.0 to a total volume of 100 uL. One ug of bovine lactoglobulin (LACB) was spiked in each sample as an internal standard. Proteins were reduced and alkylated before digestion of N-linked oligosaccharides from glycoproteins using PNGase F (Sigma-Aldrich, St. Louis, MO, USA). Proteins were then digested with trypsin and the resulting peptides were tagged with one of the isobaric tags from the iTRAQ reagents 4plex kit (AB Sciex, Foster City, CA, USA) according to the manufacturer's instructions. The four samples were then pooled and dried under vacuum. Labeled peptides were fractionated according to their isoelectric point on an Agilent 3100 OFFGEL fractionator using 24 cm pH 3-10 linear immobilized pH gradients (GE Healthcare, Chalfont St. Giles, UK) following manufacturer's instructions. Fractions were then recovered in separate tubes for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). MS analysis was performed on a LTQ Orbitrap XL from Thermo Electron (San Jose, CA, USA) equipped with a NanoAcquity HPLC system from Waters. Peptides were trapped on a home-made 5 um 200 A Magic C18 AQ (Michrom, Auburn, CA, USA) 0.1 x 20 mm pre-column and separated on a home-made 5 um 100 A Magic C18 AQ (Michrom) 0.75 x 150 mm column with a gravity-pulled emitter. The analytical separation was run for 65 min using a gradient of H_2 O/formic acid (FA) 99.9%/0.1% (solvent A) and CH_3 CN/FA 99.9%/0.1% (solvent B). The gradient was run at a flow rate of 220 nL/min as follows: 5% B for 1 min, from 5 to 35% B in 54 min, from 35 to 80% B in 10 min. For MS survey scans, the OT resolution was set to 60,000 and the ion population was set to 5 x 10^5 with an m/z window from 400 to 2000. Maximum of 3 precursors were selected for both collision-induced dissociation (CID) in the LTQ and high-energy C-trap dissociation (HCD) with analysis in the Orbitrap (OT). For MS/MS in the LTQ, the ion population was set to 1 x 10^4 (isolation width of 2 m/z) while for MS/MS detection in the OT, it was set to 2 x 10^5 , with resolution of 7,500, first mass at m/z = 100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD. Peak lists from MS analysis were searched against Uniprot_Swissprot database (version 2011_2) using Phenyx protein identification software (GeneBio, Geneva, Switzerland). /Homo Sapiens/ taxonomy was specified for database searching. The parent ion tolerance was set to 10 ppm. Variable amino acid modifications were oxidized methionine and deamidated asparagine. Carbamidomethylation of cysteines and iTRAQ-labeled peptides on the amino terminus and lysine were set as fixed modifications. Trypsin was selected as the enzyme, with one potential missed cleavage, and the normal cleavage mode was used. Only one search round was used with selection of "turbo" scoring. The peptide p value was 10^-2 for LTQ-OT data. Protein and peptide scores were set up to maintain the false positive rate below 5%.
2013-06-20 | PXD000284 | Pride
Project description:TiO2 enriched phosphopeptides of Iloprost stimulated platelets were digested with trypsin and labeled with iTRAQ. Samples were analysed by nano LC-MS/MS on an LTQ OrbitrapVelos (Thermo Scientific) coupled to an Ultimate 3000 Liquid Chromatography system (Dionex). Peptides were concentrated on a self-packed 100 µm ID reversed-phase (RP) trapping column (Kinetex C18, 2 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) in 0.1% TFA followed by separation on a self-packed 75 μm ID RP column (Kinetex C18, 30 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) using a binary gradient (solvent A: 0.1% FA and solvent B: 0.1% FA, 84% ACN) ranging from 3-45 % solvent B in 245 min at a flow rate of 230 nl/min. To minimize memory and carry-over effects from previous samples, an LC-wash program with high organic content was used prior to sample injection (Burkhart et al 2011). Survey scans were acquired in the Orbitrap with a resolution of 30,000 using the polysiloxane m/z 371.101236 as lock mass5 and MS/MS scans of the five most intense signals were acquired in the Orbitrap using HCD fragmentation (activation 0.1 ms, normalized collision energy of 50, isolation width of 1.5 m/z) with a resolution of 7,500. To compensate for elevated peptide charge states due to iTRAQ labels, a reaction tube containing 5 % ammonium water was placed in front of the ion source as described by Thingholm et. al. Database search and data processing: Raw data were processed with Proteome Discoverer 1.3 (Thermo Scientific) using Mascot (2.4) and Sequest for database search against the human Uniprot database (November, 4th 2010; 20,260 sequences) using the following search settings for both algorithms: i) trypsin with a maximum of two missed cleavages, (ii) carbamidomethylation of Cys (+57.0214 Da), peptide N-terminus iTRAQ, Lys iTRAQ (+144.102 Da) as fixed modifications and (iii) oxidation of Met (+15.9949 Da), Tyr iTRAQ (+144.102 Da) as well as phosphorylation of Ser/Thr/Tyr (+79.9663 Da) as variable modifications; (iv) MS and MS/MS tolerances of 10 ppm and 0.02 Da, respectively. A false discovery rate (FDR) of <1% was applied using the Peptide Validator node. Additionally, phosphorylation site localizations were validated by the phosphoRS node. Only phosphopeptide hits with <1% FDR of all validated peptides were considered for data interpretation and site assignment probabilities of >90 % (phosphoRS) were defined as confident. Data interpretation: To compensate for systematic variations or technical errors for each sample set, reporter areas were normalized based on modellized normal distribution with a +- 34% quantil. Therefore median iTRAQ ratios were calculated considering all unique <1% FDR peptides and normalization factors against the control sample were determined. For each peptide, all MS/MS spectra of one biological replicate were grouped in regard to modified site in protein and phosphoRS site localization (peptide groups) to determine the respective median ratios. Finally, for each peptide group, median values were calculated considering all three biological replicates. Peptides with ratios below 0.5 or above 2.0 were considered as regulated. Regulated phosphopeptides were searched against GPS 2.1 for kinase consensus site prediction. Values of high confidence (<2 % error) for PKA were taken into account.
2013-12-02 | PXD000242 | Pride
Project description:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.
2024-08-20 | PXD054722 | Pride
Project description:<p>The sample to be tested was fully ground into powder in the grinder, 50 mg of the sample was freeze-dried, 1000 μL of the extraction solution containing the inner target was added (methanol:acetonitrile:water, 2:2:1, v/v/v, internal standard concentration 20 mg/L) and vortex mixed for 30 s; then add the steel ball to the 45 Hz grinder for 10 min, ultrasonic 10 min; the sample to be tested is obtained after filtration. When detecting metabolites, metabolite determination was performed based on the LC-MS system, which mainly consists of Waters Acquity I-Class PLUS ultra-high performance liquid tandem and Waters Xevo G2-XS QTof high-resolution mass spectrometer. Meanwhile, the Waters Acquity UPLC HSS T3 column (1.8 um 2.1 x 100 mm) was used as the chromatographic column. Positive and negative ion modes were used to determine the metabolites. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% acetonitrile formate. The mobile phase conditions of liquid chromatography were as follows: the flow rate was 400 μL/min, 0.0 min: 98% flow A, 2% flow B; 0.25 min: 98% flow A, 2% flow B, 10.0 min: 2% flow A, 98% flow B; 13.0 min: 2% flow A, 98% flow B; 13.1 min: 98% flow A, 2% flow B; 15.0 min: 98% flow A, 2% flow B. MSe mode controlled by acquisition software (MassLynx v4.2, Waters) was used for primary and secondary mass spectrum data acquisition. ESI ion source parameters are as follows: Capillary voltage, 2500 V (positive ion mode) or -2000 V (negative ion mode); Cone hole voltage, 30 V; Ion source temperature, 100 °C; Desolvent temperature, 500 °C; Air flow rate, 50 L/h; Desolvent gas flow rate, 800 L/h; Plastic-nucleus ratio (m/z) collection range 50-1200 m/z. In the qualitative and quantitative analysis of metabolites, the original data collected by MassLynx v4.2 were processed by the Progenesis QI software for peak extraction, peak alignment, and other data, and the metabolites were identified based on the Progenesis QI software online METLIN database. Then, based on the results of the total score, MS2 score, and mass error (ppm), the metabolites were qualitatively determined.</p>
2024-07-12 | MTBLS10002 | MetaboLights
Project description:Expression Analysis of Bisphenol A (0.1 uM), 17beta-estradiol (0.1 uM), and GSK4716 (0.1 uM) Exposure in Embryonic Zebrafish
| PRJNA169426 | ENA
Project description:Plasma proteome response to repeated ACTH administration in juvenile northern elephant seals. Five ul of EDTA plasma were denatured with 200 ul of 5 percent w/v SDC and 5 mM TCEP in 50 mM AmBiC at 60C for 1 hour, followed by alkylation with 20 mM CAA for 30 minutes in the dark at room temp. Samples were diluted to 0.5 percent SDC and proteins were digested using trypsin at a 1 to 50 ug enzyme to protein ratio for 16 hours at 37C. TFA was added to a final concentration of 1.0 percent to precipitate SDC, which was removed by centrifugation and extraction of the supernatant. Digested peptides were lyophilized, resuspended in 0.1 percent TFA, and desalted using Pierce Peptide Desalting Spin Columns. Eluted peptides were lyophilized and resuspended in 0.1 percent FA in LC-MS grade water.
Three injections were used for each sample. For each run, 5 uL of the sample (1 ug total) was loop injected onto a reversed-phase trap column (Acclaim PepMap 100 C18 LC column; 75 um i.d. x 2 cm, 3 um particle size, 100 A pore size, Thermo Fisher Scientific, USA) by a Dionex Ultimate 3000 autosampler. Peptides were eluted onto a reversed-phase analytical column set at 35C for HPLC (EASY-SprayTM C18 LC column; 75 um i.d. x 15 cm, 100 A, Thermo Fisher Scientific, USA). Solvent A was water and B was acetonitrile (ACN), respectively (both with 0.1 percent formic acid). During each chromatographic run, which lasted 140 min, flow rates were held at 300 nl/min. Solvent B (ACN) was used as follows: 2 percent at 5 min, 2-22 percent at 75 min, 22-38 percent at 100 min, 38-95 percent at 105 min, 95 percent returning to 2 percent at 115 min, and lastly maintained at 2 percent at 140 min.
Mass spectrometry analysis was performed using Orbitrap Fusion Tribrid mass spectrometer equipped with an EASY-SprayTM ion source and operated by Xcalibur 4.0 software. MS1 spectra were resolved by the orbitrap with a resolution of 120,000, scan range of 200-1400 m/z, RF lens of 60 percent, AGC target of 1.0e6, and max injection time of 50 ms. Precursor ions were isolated by quadrupole and fragmented using HCD with a collision energy of 28 percent. MS2 product ions were resolved by the orbitrap (resolution: 30,000, AGC target: 5.0e5, first mass: 100 m/z, and max injection time: 150 ms).
2024-08-02 | MSV000095508 | MassIVE