The defatted milk proteins were electrophoretically separated using a 20% SDS-PAGE gel, and then stained with coomassie blue (Pierce, Rockford, IL). The band with size of 10 kDa was cut and sent to the Proteomics Facility at University of Vermont, where the gel pieces were digested and the extracted peptides were subjected to LC-MS/MS using a Linear Ion Trap (LTQ)-orbitrap Hybrid Mass Spectrometer (Thermo Electron, San Jose, CA).
Ltq orbitrap hybrid mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The LTQ-Orbitrap hybrid mass spectrometer is a high-performance analytical instrument designed for accurate mass measurement and advanced proteomics analysis. It combines the sensitivity and speed of a linear ion trap (LTQ) with the high resolving power and mass accuracy of an Orbitrap mass analyzer.
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Lab products found in correlation
Mascot v2, by Matrix Science (2 mentions)
Gfp trap agarose beads, by Proteintech (2 mentions)
Gelcode blue stain reagent, by Thermo Fisher Scientific (2 mentions)
Modified trypsin, by Promega (1 mentions)
Tmt10plex mass tag labeling kit, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 nanolc, by Thermo Fisher Scientific (1 mentions)
Pepmap 100 c18 trap column, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 nanolc system, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Agilent 6890 gas chromatograph, by Agilent Technologies (1 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Bioworks, by Thermo Fisher Scientific (1 mentions)
Picofrit c18 reverse phase column, by New Objective (1 mentions)
Sil 20ac autosampler, by Shimadzu (1 mentions)
500 mhz spectrometer, by Bruker (1 mentions)
Zorbax sb c18 reverse phase column, by Agilent Technologies (1 mentions)
21 protocols using ltq orbitrap hybrid mass spectrometer
1
Defatted Milk Protein Characterization
2
Comparative Quantitative Proteomic Analysis
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Bacterial proteins were extracted with 0.1% SDS solution by sonication. Forty micrograms of protein extracts were reduced and alkylated with dithiothreitol and iodoacetamide, respectively, and digested with 1 μg of modified trypsin (Promega, Madison, WI) at 37 °C overnight. Then the peptides were labeled using TMT 10 plex Mass Tag Labeling Kits (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The 2D-SCX-RP-LC experiment was performed on a Dionex Ultimate 3000 nanoflow HPLC (Dionex, Germering, Germany). The effluent of the online 2D LC was analyzed by a LTQ-Orbitrap hybrid mass spectrometer (Thermo Electron, Bremen, Germany). Raw MS files from the LTQ-Orbitrap were analyzed by Mascot v2.2.2 (Matrix Science Inc., Boston, MA) and MaxQuant v1.0.13.13.
3
Leaf Metabolite Extraction and LC-MS Analysis
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Aliquots of 200 mg of frozen, powdered leaf material were extracted with 0.6 ml of 99.9% MeOH/0.125% formic acid. After 20 s vortexing and 5 min sonication, the extracts were centrifuged for 20 min at maximum speed in a table-top centrifuge. LC-MS was performed on an Accela HPLC tower connected to a LTQ/Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific), with conditions and settings as described previously (van der Hooft et al., 2012 ).
4
Identification of BIK1 Ubiquitination Sites
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The in vitro ubiquitination reactions with GST-RHA3ACD and GST-BIK1 or GST-BIK1K204R were performed as mentioned above with overnight incubation. Reactions were loaded on an SDS-PAGE gel (7.5%) and ran for a relatively short time untill the ubiquitinated bands can be separated from the original GST-BIK1 (GST-BIK1 band ran less than 0.5 cm from the separating gel). Ubiquitinated bands were sliced, trypsin digested before LC-MS/MS analysis on an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher) as previously described30 (link). The MS/MS spectra were analyzed with SEQUEST software, and images were exported from SEQUEST.
In vivo BIK1 ubiquitination sites were identified as following: 20 ml of WT arabidopsis protoplasts with a concentration of 2 ×105 per ml were transfected with BIK1-GFP and FLAG-UBQ and the protoplasts were treated with 200nM flg22 for 30 min after 7 hr incubation. GFP-trap-Agarose beads (Chromotek, Cat # gta-20) were incubated with cell lysates in the ratio of 10 μl beads per 4 ×105 cells for 1 hr at 4°C and beads were pooled from 10 tubes, washed using IP buffer for 3 times, and denatured in SDS buffer. Samples were separated by 10% SDS-PAGE and stained with GelCode Blue Stain Reagent (Thermo Fisher Cat # 24590). Ubiquitinated bands were sliced and analyzed as mentioned above.
In vivo BIK1 ubiquitination sites were identified as following: 20 ml of WT arabidopsis protoplasts with a concentration of 2 ×105 per ml were transfected with BIK1-GFP and FLAG-UBQ and the protoplasts were treated with 200nM flg22 for 30 min after 7 hr incubation. GFP-trap-Agarose beads (Chromotek, Cat # gta-20) were incubated with cell lysates in the ratio of 10 μl beads per 4 ×105 cells for 1 hr at 4°C and beads were pooled from 10 tubes, washed using IP buffer for 3 times, and denatured in SDS buffer. Samples were separated by 10% SDS-PAGE and stained with GelCode Blue Stain Reagent (Thermo Fisher Cat # 24590). Ubiquitinated bands were sliced and analyzed as mentioned above.
5
LC-MS/MS Analysis of Peptides
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For LC-MS/MS analysis, we used a reversed-phase liquid chromatography (RP-LC) system (Ultimate 3000 nano LC) interfaced with an LTQ-Orbitrap hybrid mass spectrometer equipped with a nanoelectrospray ion source (all from Thermo Fisher Scientific). The RP-LC system comprised a C18 PepMap 100 trap column (length × inner diameter: 0.5 × 0.3 mm; Thermo Fisher Scientific) and a C18 tip column (length × inner diameter: 10 cm × 75 μm; particle diameter: 3 μm; Nikkyo Technos, Tokyo, Japan). Samples were loaded onto the trap column, washed with H2O containing 0.1% formic acid (solvent A) to concentrate and desalt them, and eluted using 95% acetonitrile, 5% H2O and 0.1% formic acid (solvent B). The 120-min LC gradient changed from 97.5% A/2.5% B to 77.5% A/22.5% B at 109 min, 65% A/35% B at 5 min, and finally 2% A/98% B at 2 min (at 0.3 μL/min). Eluted peptides were ionized by the electrospray and analyzed using the mass spectrometer (electrospray voltage: 1.8 kV; no sheath and auxiliary gas flow; capillary temperature: 250°C; collision energy: 35%; ion-selection threshold: 500 counts for MS/MS; Top N: 15; dynamic exclusion: 60 s).
6
Proteome Analysis by nanoLC-MS/MS
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Samples were analyzed using a Dionex Ultimate 3000 nanoLC system (Thermo Scientific) coupled to an LTQ-Orbitrap hybrid mass spectrometer (ThermoFisher Scientific, Bremen, Germany). Chromatographic separations were performed on a reversed-phase capillary column (75 μm × 15 cm, particle size 1.7 μm, pore size 15 nm). Then the peptides in 0.1% FA were separated using a solvent gradient of increasing from 3% to 30% solvent B (0.1% FA in 98% acetonitrile) over 100 min at a flow rate of 300 nL/min.
Data-dependent acquisition in positive mode recorded MS scans in profile mode from m/z 300–2000. The 20 most intense precursor ions were selected for MS2 collision induced dissociation fragmentation with an isolation window of 2.0 Da and dynamic exclusion set at 10.0 s. Automatic gain control targets of 5E4 were accumulated for MS/MS spectra generation.
Data-dependent acquisition in positive mode recorded MS scans in profile mode from m/z 300–2000. The 20 most intense precursor ions were selected for MS2 collision induced dissociation fragmentation with an isolation window of 2.0 Da and dynamic exclusion set at 10.0 s. Automatic gain control targets of 5E4 were accumulated for MS/MS spectra generation.
7
High-throughput MS/MS Datasets from Diverse Cell Lines
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An MS/MS dataset from 11 human cell lines (A549, GAMG, HEK293, HeLa, HepG2, Jurkat, K562, LnCap, MCF7, RKO, and U2OS, each with three replicates) was obtained using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) [9 (link)]. The HEK293 24-fraction MS/MS dataset was obtained with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) [10 (link)]. The S. cerevisiae Elite MS/MS dataset was obtained with an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) [11 (link)]. The S. cerevisiae 2DLC MS/MS dataset was obtained using a LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) [12 (link)]. Peptide fragmentation was performed using the higher-energy collisional dissociation (HCD) method. Supplementary Table 1 shows the number of spectrum in the human cell lines, the HEK293 24 fraction, in the S. cerevisiae Elite and 2DLC datasets.
8
Comprehensive Metabolite Profiling of Fruit Samples
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Volatile compounds were quantified and identified using headspace solid‐phase micro‐extraction–gas chromatography–mass spectrometry (SPME‐GC‐MS), as described by Tikunov et al. (2005 ).
Soluble solid content (SSC) and acidity of the fruit material were analysed using a portable handheld PAL BX/ACID3 analyzer (Atago,https://www.atago.net ). Frozen fruit powder was thawed at 22ºC room temperature, centrifuged and the juice was subjected to analyses according the analyzer manual.
Individual sugars, and organic and amino acids, were analysed using an Agilent 6890 gas chromatograph (https://www.agilent.com ) coupled to a Pegasus III time‐of‐flight (TOF) mass spectrometer (LECO, https://www.leco.com ) using the sample preparation and instrumental methods described previously by Osorio et al. (2012 ) and Carreno‐Quintero et al. (2012 ), respectively.
Semi‐polar secondary metabolites were analysed using the LTQ Orbitrap LC‐MS system composed of a HyPurityTM C18 column (ThermoFisher Scientific,https://www.thermofisher.com ), an Acquity UPLC coupled to a photodiode array (PDA) detector (both from Waters, https://www.waters.com ) and an LTQ/Orbitrap hybrid mass spectrometer (ThermoFisher Scientific), as previously described by van der Hooft et al. (2012 ).
Soluble solid content (SSC) and acidity of the fruit material were analysed using a portable handheld PAL BX/ACID3 analyzer (Atago,
Individual sugars, and organic and amino acids, were analysed using an Agilent 6890 gas chromatograph (
Semi‐polar secondary metabolites were analysed using the LTQ Orbitrap LC‐MS system composed of a HyPurityTM C18 column (ThermoFisher Scientific,
9
Quantitative Analysis of Regorafenib Metabolites
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Selected plasma and fecal extracts, urine, and isolated metabolites M-7 and M-8 (see Online Resource, Appendix A) were analyzed on an HP 1200 HPLC system (Hewlett-Packard, Waldbronn, Germany) coupled to a LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany) equipped with heated electrospray ionization (HESI_2) interface. Structures of metabolites in excreta were confirmed by HPLC–HRMS, nuclear magnetic resonance analysis, and by chromatographic/LC–HRMS/MS comparison with authentic reference samples (M-2 to M-6). The metabolite structures are shown in Table 1 .
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Assignment and proposed structures of the metabolites of regorafenib identified in in vitro incubations and in vivo studies (for further information see Online Resource Appendix C)
10
HLA-A*02 Peptide Profiling of Tumor Samples
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Nine HLA-A*0201+ tumor biopsies samples were analysed and compared to a panel of 244 non-tumoral body tissue including 7 brain tissues. Shock-frozen tumor samples were essentially processed as described previously47 according to standard protocols.48 (link) Briefly, HLA-A*02 peptide pools from shock-frozen tissue samples were obtained by immune precipitation from solid tissue using HLA-specific antibodies, acid treatment and ultrafiltration. To obtain samples containing HLA-A*02-restricted peptides the antibody BB7.2 was used.49 The HLA peptide pools as obtained were separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPLC system, Waters) and the eluted peptides were analysed in an LTQ Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) equipped with an electrospray ionization source. The LC–MS data were collected and automatically processed by analyzing the LC–MS survey (mass signals of unfragmented peptides) as well as the tandem-MS (MS/MS) data (fragment spectra containing peptide sequence information). Automated data analysis had been optimized and adapted for identification and quantification of HLA-restricted peptides.
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