Approximately 2 μg of tryptic digest for each sample was injected using an EASY nanoLC-II system (Thermo Scientific, San Jose, CA). Peptides were purified and concentrated using an online enrichment column (EASY-Column, 100 μm ID × 2 cm ReproSil-Pur C18). Subsequent chromatographic separation was performed on a reverse phase nanospray column (EASY-Column, 3μm, 75 μm ID × 100 mm ReproSil-Pur C18) using a 90 min linear gradient from 10%–35% buffer B (100% ACN, 0.1% formic acid) at a flow rate of 400 nL/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific Orbitrap Velos). The instrument was operated in Orbitrap-LTQ mode where precursor measurements were acquired in the Orbitrap (60,000 resolution) and MS/MS spectra (top 20) were acquired in the LTQ ion trap with a normalized collision energy of 35%. Mass spectra were collected over a m/z range of 400–2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific) with a S/N threshold of 1.5 and 1 scan/group.
Easy nano lc 2 system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Easy Nano LC II system is a liquid chromatography instrument designed for the separation and analysis of nanoscale samples. It enables high-resolution separation and sensitive detection of compounds in complex matrices. The system is equipped with a low-flow, high-pressure pump and an integrated autosampler for automated sample handling.
Automatically generated - may contain errors
Lab products found in correlation
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (3 mentions)
Orbitrap velos, by Thermo Fisher Scientific (2 mentions)
C18 pepmap100, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap elite, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer, by Thermo Fisher Scientific (2 mentions)
Proteomass ltq ft hybrid esi positive mode cal mix mscal5 supelco, by Merck Group (2 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions)
Xcalibur 2, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap velos pro, by Thermo Fisher Scientific (1 mentions)
Nanospray flex, by Thermo Fisher Scientific (1 mentions)
Magic c18aq, by Bruker (1 mentions)
Reprosil pur c18 aq, by Thermo Fisher Scientific (1 mentions)
Nano bore stainless steel online emitter, by Thermo Fisher Scientific (1 mentions)
C18aq analytical column, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (1 mentions)
Trypsin gold, by Promega (1 mentions)
Xcalibur version 2.1 software, by Thermo Fisher Scientific (1 mentions)
Trypsin lys c, by Promega (1 mentions)
14 protocols using easy nano lc 2 system
1
Nano-LC MS/MS Proteomics Protocol
2
Cross-linking and Mass Spectrometry Analysis of MBP-Nop58p_447-R2TP Complex
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The cross-linking reaction and mass spectrometry were performed similarly as described in ref. 28 with exception for the sample used. Briefly, the co-purified MBP-Nop58p_447-R2TP complex analyzed by cryoEM was incubated with 0.5% deuterated (d4) or nondeuterated (d0) bis(sulfosuccinimidyl)suberate (BS3) for 30 min. The BS3-d0 and BS3-d4 cross-linked samples were mixed in equal volume, purified on a 7.5% SDS-PAGE gel, and digested by trypsin for mass spectrometry. Digested peptides were separated on an Easy Nano LC II system (Thermo Fisher Scientific), ionized by electrospray ionization (ESI) and detected by a Velos LTQ-Orbitrap Mass Spectrometer (Thermo Scientific). Cross-linked peptides showed the presence of both BS3-d0 and BS3-d4 cross-linkers, ion pairs separated by 4.0247 Da and were verified mannually.
3
Reverse-Phase LC-MS/MS Fractionation and Analysis
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Fractions were separated by on-line reversed phase liquid chromatography using a Thermo Scientific EASY-nanoLC II system with a reversed-phase pre-column Magic C-18AQ (100 μm I.D., 2 cm length, and an in-house prepared reversed-phase nano-analytical column packed with Magic C-18AQ (75 μm I.D., 15 cm length, 5 μm, 100 Å; Michrom BioResources Inc, Auburn, CA). The chromatography system was coupled on-line to an LTQ Orbitrap Velos Pro mass spectrometer equipped with a Nanospray Flex source (Thermo Fisher Scientific, Bremen, Germany) and run over a 120 min gradient from 95 % solvent A (2 % Acetonitrile, 0.1 % Formic acid):5 % solvent B (90 % Acetonitrile, 0.1 % Formic acid) to 100 % solvent B. Mass spectrometry data were acquired with a time of flight survey scan of mass range 400–1800 amu where the most abundant ions exceeding 5000 counts and charge state 2–4 selected for fragmentation.
4
Peptide Fractionation and LC-MS/MS Analysis
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For each sample, peptide fractions were solubilized in FA 0.1% (v/v) and analyzed on a LTQ-Orbitrap Elite apparatus coupled to an Easy nanoLC II system (Thermo Scientific). An amount of 0.2 µg of peptides was injected onto an enrichment column (C18 Pepmap100, Thermo Scientific). The separation was carried out with an analytical column needle (NTCC-360/100-5-153, Nikkyo-Technos). The flow rate was 300 nL/min and the mobile phase composed of H2O/0.1% FA (buffer A) and ACN/0.1% FA (buffer B). The elution gradient duration was 120 min: 0-106 min, 2–40% B; 106-110 min, 40–100% B; 110-120 min, 100% B. The mass spectrometer was operated in positive mode with CID fragmentation. For mass spectrometry settings, the capillary voltage was 1.5 kV and the temperature of the capillary was 275 °C. The m/z detection range was 400–1800 in MS scan at a resolution of 60 000. The 20 most intense peptide ions were selected and the fragmentation occurred with a normalized collision energy of 35. Dynamic exclusion of already fragmented precursor ions was applied for 30 s.
5
Proteomic Analysis of Gel-digested Samples
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The gel band (from IP result) was eventually digested by the in-gel trypsin digestion process, following previous procedures36 (link). Peptide samples were analyzed on an LTQ-Orbitrap Velos (Thermo Fisher Scientific) connected to an Easy-nano LC II system (Thermo Fisher Scientific) incorporated with an autosampler. The dried peptide samples were resuspended in 70 μL of 0.1% formic acid, and an aliquot (7 μL) was injected into a reversed-phase peptide trap EASY-Column (L 2 cm, ID 100 μm, 5 μm, 120 Å, ReproSil-Pur C18-AQ, Thermo Fisher Scientific) and a reversed-phase analytical EASY-Column (L 10 cm, ID 75 μm, 3 μm, 120 Å, ReproSil-Pur C18-AQ, Thermo Fisher Scientific), and electrospray ionization was subsequently performed using a 30 μm (i.d.) nano-bore stainless steel online emitter (Thermo Fisher Scientific). The total duration of LC gradient analysis was 60 min. The peptides were eluted in a linear gradient of 10–40% buffer B over a period of 40 min, with buffer A (0.1% formic acid in H2O) and buffer B (0.1% formic acid in acetonitrile), and a flow rate of 0.3 μL/min. The temperature and voltage applied to the capillary was 275 °C and 1.9 V, respectively. All data were acquired with the mass spectrometer operating in automatic data-dependent switching mode. The MS survey was scanned from 350 to 2000 m/z with the resolution set to 100,000.
6
Proteomic Analysis of Yeast 90S Ribosome
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Purified 90S was subjected to mass spectrometry analysis for protein identifications. Trypsin digested 90S sample was injected for nLC-MS/MS analysis (Easy Nano LC II system and a Velos LTQ-Orbitrap Mass Spectrometer, Thermo Scientific). Peptides were separated with a 10 cm × 75 µm C18AQ analytical column (Thermo Scientific). Peptide/protein identification was conducted by searching against a Uniprot S. Cerevisiae database with Proteome Discoverer 1.4 (Thermo Scientific) Sequest HT search engine (University of Washington). The precursor ions mass error tolerance is <5 ppm and fragment ions mass error tolerance is <0.8 Da and Percolator at a 1% FDR. Search parameters were as follows: trypsin, allowing 4 miscleavages, with oxidized methionine and carbamidomethyl cysteine as dynamic modifications. High confidence proteins are listed in Supplementary Data 1 .
7
Ubiquitinated KRAS4B Proteomics Analysis
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Strep-DX2 was introduced into 293T cells expressing GFP-KRAS4B. Cells treated with MG-132 and KRAS4B proteins were purified by immunoprecipitation with an anti-GFP antibody (n = 1). Equivalent amounts of eluted protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. KRAS4B proteins separated by SDS-PAGE were subjected to in-gel digestion with trypsin GOLD (Promega). Analysis of the peptide mixture was performed using LTQ-Orbitrap Velos (Thermo Fisher Scientific) connected to Easy-nano LC II system (Thermo Fisher Scientific) with an incorporated autosampler. Acquired data were analyzed from the data-dependent mode to simultaneously record full-scan mass and collision-induced dissociation (CID) spectra with multistage activation. Mass spectra were searched against the KRAS4B sequence database (Uniprot accession No.: P01116-2) using Proteome Discoverer (version 1.3, Thermo Scientific, Waltham, MA USA) with the SEQUEST search engine. The precursor mass tolerance and fragment mass tolerance were set to 25 ppm and 0.8 Da. For ubiquitinated peptide identification, lysine ubiquitination (+114.04 Da) and methionine oxidation (+15.99 Da) were set as variable modifications and cysteine carbamidomethylation (+57.02 Da) was set as a static modification.
8
MS Analysis of Dietary Restriction Proteins
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DRPs were analysed on EASY nanoLC II system coupled with LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), previously calibrated with the ProteoMass™ LTQ/FT-Hybrid ESI Positive Mode Cal Mix (MSCAL5 SUPELCO, Sigma-Aldrich) calibration set. Samples were also analysed under reducing and alkylating condition. Reduction and alkylation of the DRPs was done as previously described for peanut conglutins17 (link). Two microliters (concentration of 50 μg/ml) of each sample was injected onto the trapping column (EasyColumn C18, 2 cm length, ID 100 μm, 5 μm particle size) and separation was performed on an Easy spray PepMap C18, (length 15 cm, ID 75 μm, particle size 3 μm) as described17 (link). Spray was generated with a stainless steel emitter, with tip voltage set at 2.35 kV, capillary voltage 6 V and capillary temperature of 275 °C. A high-resolution full Fourier-Transform Mass Spectrometry (FTMS) profile spectrum (scan range 300–3000 m/z, resolving power 60 000, 1 microscan) was acquired using Xcalibur (version 2.1) software (Thermo Fisher Scientific) with the precursor mass tolerance of 10 ppm. The experiments were done in duplicate.
9
Peptide Fractionation and LC-MS/MS Analysis
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For each sample, peptide fractions were solubilized in FA 0.1% (v/v) and analyzed on a LTQ-Orbitrap Elite apparatus coupled to an Easy nanoLC II system (Thermo Scientific). Peptides were injected onto an enrichment column (C18 Pepmap100, Thermo Scientific). The separation was carried out with an analytical column needle (NTCC-360/100-5-153, Nikkyo-Technos). The flow rate was 300 nL/min and the mobile phase composed of H2O/0.1% FA (buffer A) and ACN/0.1% FA (buffer B). The elution gradient duration was 120 minutes: 0-106 min, 2-40% B; 106-110 min, 40-100% B; 110-120 min, 100% B. The mass spectrometer was operated in positive mode with CID fragmentation. For mass spectrometry settings, the capillary voltage was 1.5 kV and the temperature of the capillary was 275°C. The m/z detection range was 400-1800 in MS scan at a resolution of 60 000. The 20 most intense peptide ions were selected and the fragmentation occurred with a normalized collision energy of 35. Dynamic exclusion of already fragmented precursor ions was applied for 30 seconds.
10
AAGAB Protein Crosslinking and Identification
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The purified AAGAB protein was crosslinked with a solution of 1:1 BS3-d0: BS3-d4 (ThermoFisher #21590 and #21595) crosslinkers. Crosslinking reaction product was separated by SDS-PAGE. The bands corresponding to dimer and tetramer were cut. Cut gel bands were destained, reduced with dithiothreitol, and digested with trypsin at 37 °C overnight. Tryptic peptides were separated by an Easy Nano LC II system (Thermo Scientific). Mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A 3-h gradient (from 5 to 45% B) was performed with a flow rate of 300 nL/min. nLC_eluates were online ionized by nano-electrospray ionization and detected by a Velos LTQ-Orbitrap Mass Spectrometer (Thermo Scientific). Precursor ions were detected in the Orbitrap with a mass resolution of 60 K, while the data-dependent MS2 of the top 10 most abundant precursor ions were carried out in LTQ. The collected .raw files were converted to .mzXML files for crosslinking data analysis with StavroX, an open-access software (34 (link)). Search parameters were used as followed: max 4 trypsin miscleavages, dynamic modification of methionine by oxidation, precursor mass accuracy of better than 5 ppm, and fragment ion mass accuracy of better than 0.8 Da. StavroX generated crosslinked peptide list was further manually checked for assignment.
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