Dried peptides were reconstituted with 2% acetonitrile, 0.1% formic acid, and quantified by modified BCA peptide assay (Thermo Fisher Scientific). Equal peptide amounts derived from each sample were injected and analyzed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Acclaim PepMap column (0.075 × 500 mm, 2 µm; Thermo Scientific) equilibrated with buffer A (0.1% formic acid in water) and eluted in a 93-min linear gradient of 2–28% solvent B (100% acetonitrile) at a flow rate of 300 nL/min. The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured with a resolution of 70,000, an automated gain control (AGC) target of 1e6 and a mass range from 350 to 1700 m/z. Up to 12 MS2 spectra per duty cycle were triggered, fragmented by higher energy collisional dissociation (HCD), and acquired with a resolution of 17,500 and an AGC target of 5e4, an isolation window of 1.6 m/z and a normalized collision energy of 25. Dynamic exclusion was enabled with duration of 20 s.
Proxeon easy nanolc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Proxeon EASY nanoLC system is a high-performance liquid chromatography (HPLC) instrument designed for nanoscale separations. It provides precise and reproducible sample delivery for sensitive liquid chromatography-mass spectrometry (LC-MS) applications.
Automatically generated - may contain errors
Lab products found in correlation
Acclaim pepmap c18 column, by Thermo Fisher Scientific (3 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophometer, by Thermo Fisher Scientific (2 mentions)
Data analysis software, by Bruker (1 mentions)
Microtof q 2, by Bruker (1 mentions)
Nupage gradient gel, by Thermo Fisher Scientific (1 mentions)
Hct ultra ion trap mass spectrometer, by Bruker (1 mentions)
Mascot, by Matrix Science (1 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x, by Thermo Fisher Scientific (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Trypsin lys c mix, by Promega (1 mentions)
10 protocols using proxeon easy nanolc system
1
LC-MS/MS Peptide Quantification Protocol
2
Liquid Chromatography-Mass Spectrometry Proteomics
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Dried peptide fractions were reconstituted with 2% ACN-0.1% FA and analyzed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Acclaim PepMap column (75μm x 250 mm, 2μm particles, Thermo Scientific) at a flow rate of 300 μl/min using a 58-min gradient: 1% to 6% B in 1 min, 6% to 23% B in 35 min, and 23% to 34% B in 22 min (A = FA, 0.1%; B = 80% ACN: 0.1% FA). The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured with a resolution of 70,000 (AGC target: 1e6; mass range: 350–1700 m/z). Up to 12 MS2 spectra per duty cycle were triggered, fragmented by HCD, and acquired with a resolution of 17,500 (AGC target 1e5, isolation window; 1.2 m/z; normalized collision: 32) Dynamic exclusion was enabled with a duration of 25 sec.
3
Nano-LC-MALDI-TOF/TOF Proteomic Analysis
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The equal amount of peptides in each fraction was injected into the nNano-liquid chromatography (Nano-LC) system. For analysis using MALDI-TOF/TOF, the SCX peptide fractions were pooled to reduce peptide complexity to yield 17 fractions. A 10-μl portion from each fraction was injected twice into the Proxeon Easy Nano-LC system (Thermo Scientific, West Palm Beach, Florida, USA). Peptides were separated on a C18 analytical reverse phase column at a flow rate of 300 nl/min with solvent (solution A, 5% CAN and 0.1% formic acid; solution B, 95% ACN and 0.1% formic acid) for 120 min. A linear LC gradient profile was used to elute peptides from the column, commencing with 5% solution B. After equilibration in 5% solution B, a multi-slope gradient started 10 min after the injection signal as follows: 45% solution B for 80 min; 80% solution B for 15 min; 5% solution B for 15 min; and then 5% solution B. Fractions were analyzed using a hybrid quadrupole/time-of-flight MS (Micro TOF-Q II; Bruker, Bremen, Germany) with nano electrospray ion source. Data were collected and analyzed using Data Analysis Software (Bruker). Nitrogen was used as the collision gas, and the MS/MS scans from 50–2,000 m/z were recorded. The ionization tip voltage and interface temperature were set at 1,250 V and 150°C, respectively.
4
LC-MS/MS Analysis of Peptides
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Prior to LC-MS/MS analysis, dried peptides were reconstituted with 2% acetonitrile (ACN) and 0.1% formic acid (FA), and concentration was determined using a NanoDropTM spectrophometer (ThermoFisher). Samples were then analyzed by LC-MS/MS using a Proxeon EASY-nanoLC system (ThermoFisher) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Aurora column (75 µm × 250 mm, 1.6 µm particles; IonOpticks) at a flow rate of 300 nL/min (60 C) using a 120 min gradient: 1% to 5% B in 1 min, 6% to 23% B in 72 min, 23% to 34% B in 45 min, and 34% to 48% B in 2 min (A=FA 0.1%; B=80% ACN: 0.1% FA). The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured in the Orbitrap in a mass-to-charge (m/z) of 350–1700 with a resolution of 70,000 at m/z 400. Automatic gain control target was set to 1×106 with a maximum injection time of 100ms. Up to 12 MS2 spectra per duty cycle were triggered, fragmented by higher-energy C-trap dissociation (HCD), and acquired with a resolution of 17,500 and an AGC target of 5×104, an isolation window of 1.6 m/z, and a normalized collision energy of 25. The dynamic exclusion was set to 20 s with a 10 ppm mass tolerance around the precursor.
5
Peptide Separation and Identification by LC-MS/MS
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Dried samples were reconstituted with 2% acetonitrile, 0.1% formic acid and analyzed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Acclaim PepMap column 0.075 × 250 mm, 2 µm particles (Thermo Scientific) in a 180-min gradient of 2–28% solvent B at a flow rate of 300 nL/min. The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured with a resolution of 60,000, an AGC target of 1e6 and a mass range from 350 to 1400 m/z. Up to 10 MS2 spectra per duty cycle were triggered, fragmented by collision-induced dissociation and acquired in the ion trap with an AGC target of 1e4, an isolation window of 2.0 m/z and a normalized collision energy of 35. Dynamic exclusion was enabled with duration of 30 sec.
6
Proteomic Identification of Peptides
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Samples for mass spectrometry were separated on 4%–12% gradient NuPAGE gels (Invitrogen, Carlsbad, CA, United States), followed by colloidal Coomassie blue staining. Gel lanes were cut into 14 slices and then digested with trypsin. Peptides were separated using the Proxeon EasyNanoLC system (Thermo Fisher, Waltham, MA, United States) fitted with a trapping column (Hydro-RP C18 (Phenomenex, Torrance, CA, United States), 100 μm × 2.5 cm, 4 μm) and an analytical column (Reprosil C18, 75 μm × 15 cm, 3 μm, 100 Å). The outlet of the analytical column was coupled directly to an HCT Ultra Ion Trap mass spectrometer (Bruker Daltonics, Billerica, MA, United States) using the ESI nanoflow source in positive ion mode. Peptides were identified via Mascot (Matrix Science, London, United Kingdom) using the Swiss-Prot database.
7
Sensitive LC-MS/MS Proteomic Analysis
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Dried samples were reconstituted with 2% acetonitrile, 0.1% formic acid and analyzed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Acclaim PepMap column 0.075 × 500 mm, 2 μm particles (Thermo Scientific) in a 90-min linear gradient of 2–28% solvent B at a flow rate of 300 nL/min. The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured with a resolution of 120,000, an AGC target of 1e6, a maximum injection time of 100 ms and a mass range from 350 to 1400 m/z. The instrument was set to run at top speed mode with 3 s cycles for the survey and the MS/MS scans. After a survey scan, tandem MS was performed on the most abundant precursors exhibiting a charge state from 2 to 8 of greater than 5e3 intensity by isolating them in the quadrupole at 0.8 Th. HCD fragmentation was applied with 30% collision energy and the resulting fragments were detected using the turbo scan rate of the ion trap. The AGC target for MS/MS was set to 1e4 and the maximum injection time limited to 15 ms. The dynamic exclusion was set to 15 s with a 10 ppm mass tolerance around the precursor and its isotopes.
8
Proteomic Analysis of Dried Samples
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Dried samples were reconstituted with 2% ACN-0.1% FA and quantified by NanoDrop spectrophometer (Thermo Fisher Scientific) prior to LC-MS/MS analysis using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Acclaim PepMap column (75 μm x 250 mm, 2 μm particles; Thermo Fisher Scientific) using a 117-min gradient, at a flow rate of 300 μl/min, consisting in: 1% to 6% B in 1 min, 6% to 23% B in 72 min, 23% to 34% B in 45 min, 34% to 48% B in 2 min, and 48% to 98% B in 2 min (A = FA, 0.1%; B = 80% ACN: 0.1% FA). The MS was operated in positive data-dependent acquisition mode. MS1 spectra were measured in the Orbitrap with a resolution of 60,000 (AGC target: 3e4; maximum injection time: 100 ms; mass range: from 375 to 1400 m/z). After the survey scan, up to 10 of the most intense precursors ions were fragmented by CID in the ion trap cell (Isolation window: 2 m/z; charge state: + 2; normalized collision energy: 35%). Resulting fragments were detected in the Ion trap cell with a rapid scan (AGC target: 1e4; maximum injection time: 100 ms). Precursor dynamic exclusion was set to 30s, with a 10 ppm mass tolerance around the precursor.
9
Peptide Analysis via Nano-LC-MS/MS
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Peptides were reconstituted in buffer LC-A (2% ACN, 0.1% FA) and analyzed with a Proxeon EASY-nanoLC system (Thermo Scientific) interfaced to a Q-Exactive HF-X (for proteomics) or Q-Exactive HF (for metabolomics) mass spectrometers (Thermo Scientific) through a nanoEASY source (Thermo Scientific). Peptides were resolved on a PepMap RSLC C18 analytical column (2 μm beads, 50 μm internal diameter, 50 cm long) separated from nanoLC by an Acclaim PepMap 100 C18 nanoViper trap (3 μm beads, 75 μm internal diameter, 2 cm long). We used a flow rate of 200 nL/min and an increasing gradient of buffer LC-B (80% ACN, 0.1% FA). The spectra were acquired using the XCalibur software (Thermo).
10
Proteomics Analysis of Immunoprecipitated Samples
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Following immunoprecipitation, proteins were digested overnight directly on-beads using mass spec grade Trypsin/Lys-C mix (Promega, Madison, WI) following cysteine reduction with 10 mM tris(2-carboxyethyl) phosphine (TCEP) and alkylation with 30 mM iodoacetamide (IAA). The digested samples were desalted using a C18 TopTip (PolyLC, Columbia, MD). Peptide samples were then analysed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Acclaim PepMap column 0.075 × 500 mm, 2 µm particles (Thermo Scientific) in a 90-min gradient of 2–28% solvent B at a flow rate of 300 nL/min. The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured with a resolution of 60,000, an AGC target of 1e6 and a mass range from 350 to 1400 m/z. Up to 10 MS2 spectra per duty cycle were triggered, fragmented by CID and acquired in the ion trap with an AGC target of 1e4, an isolation window of 2.0 m/z and a normalised collision energy of 35. Dynamic exclusion was enabled with duration of 30 sec.
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