Samples were measured on a nanoLC coupled via nano electrospray ionization to the QExactive HF‐x benchtop instrument by the Proteomics Facility of the CECAD Institute (Cologne). In brief, samples were analyzed after in‐solution digestion using a 90 min gradient with linearly increasing concentrations of solvent B for 65 min, followed washing at 95% B and 5 min re‐equilibration to 5% solvent B on a 50 cm column (i.d. 75 μm), packed in house with 1.9 μm C18 beads (Dr. Maisch, Germany). For label free quantitation, spectra were acquired at a resolution of 70 000 at 200 m/z. An automated gain control (AGC) target of 3 x 106 and a maximum injection time of 20 msec were used. Tandem mass spectrometry (MS/MS) spectra were acquired in a top 22 data‐dependent mode using a resolution of 15 000 at 200 m/z after accumulation of 5 × 105 AGC targets within an injection time of 20 ms. Ions were isolated at a 1.3 Th isolation window and fragmented in the HCD cell at 27 normalized energy. Dynamic exclusion was set to 25 sec.
C18 beads
Manufactured by Dr. Maisch
Sourced in Germany
C18 beads are a type of chromatography media used for separation and purification of various molecules. They consist of silica particles coated with C18 alkyl chains, which provide a reversed-phase interaction mechanism for retaining and separating analytes. C18 beads are commonly used in high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) applications.
Automatically generated - may contain errors
Lab products found in correlation
Easy nlc 1200, by Thermo Fisher Scientific (5 mentions)
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Q exactive hf, by Thermo Fisher Scientific (2 mentions)
Q exactive hf x, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000 ultra high pressure system, by Thermo Fisher Scientific (1 mentions)
Nano electrospray source, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Nanoacquity uplc, by Thermo Fisher Scientific (1 mentions)
Complete protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Microcon filter, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Orbitrap eclipse tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easylc 1200, by Thermo Fisher Scientific (1 mentions)
Dextrose, by Thermo Fisher Scientific (1 mentions)
Difco yeast nitrogen base, by BD (1 mentions)
Formic acid, by Merck Group (1 mentions)
Reprosil aq pur, by Dr. Maisch (1 mentions)
23 protocols using c18 beads
1
Nanoflow LC-MS/MS Proteomics Protocol
2
Trypsin and Elastase Digestion Protocol
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Trypsin and elastase digests for each sample were resuspended with 25 µL 80% acetonitrile (ACN) and combined. A total of 40 µL was transferred to a 96-well plate prior to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. To remove the ACN, the plate was vacuum centrifuged until ∼5 µL remained. Samples were then resuspended with 8 µL 0.1% TFA 5% can, and 5 µL was then separated by an EASY-nLC 1200 (Proxeon) attached to a Q-Exactive HF-X mass spectrometer (Thermo Scientific) on a 15-cm column (75 μm inner diameter; made in-house laser pulled and packed with 1.9-μm C18 beads [Dr. Maisch] over a 77-min gradient). The parameters were the same as those already published for historical samples (99 (link)). In short, MS1: 120k resolution, maximum injection time (IT) 25 ms, scan target 3E6. MS2: 60k resolution, top 10 mode, maximum IT 118 ms, minimum scan target 3E3, normalized collision energy of 28, dynamic exclusion 20 s, and isolation window of 1.2 m/z. A wash-blank consisting of 0.1% TFA 5% ACN was run before and after each sample to hinder cross-contamination.
3
Larval Proteomics: Temperature Influence
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Data-dependent acquisition was performed on an Orbitrap Fusion Lumos Mass Spectrometer (Thermo Scientific) at the University of Washington Proteomics Resource to assess the effect of temperature on proteomic profiles throughout larval development. Technical duplicates for each sample were processed by liquid chromatography coupled to tandem mass spectrometry (LC − MS/MS). Briefly, the analytical column (20 cm long) was packed in house with C18 beads (Dr. Maisch HPLC, Germany, 0.3 μm) with a flow rate of 0.3 μL/min. Chromatography was carried out with an increasing ratio of acetonitrile + 0.1% formic acid (solvent A):water + 0.1% formic acid (solvent B). The solvent gradient was 5–95% solvent A over 70 min. Quality-control standards (Pierce Peptide Retention Time Calibration mixture + bovine serum albumin) were analyzed throughout the experiment to ensure consistency of peptide detection and elution times.
4
Trypsin-Based Peptide Preparation for MS
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After the last IP wash, proteins were on-bead digested by trypsin, as previously described (29 (link)). Peptides were desalted on 2xC18 StageTips. Tryptic peptides were separated on an in-house packed 15-cm column (inner diameter, 75 µm, 1.8 µm C18 beads; Dr. Maisch GmbH) using the Easy nano-flow high-performance liquid chromatography system. Peptides were loaded on the column in buffer A (0.5% formic acid) and eluted with a 100-min linear gradient from 2 to 40% buffer B (80% acetonitrile, 0.5% formic acid). The high-performance liquid chromatography system was coupled via a nano electrospray ion source to a Q Exactive HFX Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). Mass spectra were generated by the data-dependent acquisition with automatic switching between MS and tandem MS (MS/MS). Precursor ions were picked using the top-15 method. Tandem spectra were generated using high collision dissociation (27 normalized collision energy). MS and MS/MS spectra were acquired at 60,000 and 15,000 resolutions respectively using the Orbitrap analyzer.
5
LC-MS/MS Proteomics Analysis Protocol
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MS-based proteomic measurements were performed as in ref. 26 (link). Briefly, ~2 µg of desalted peptides were loaded and analyzed by linear 4 h gradients. The LC system was equipped with an in-house made 50-cm, 75-µm inner diameter column slurry-packed into the tip with 1.9 µm C18 beads (Dr. Maisch GmbH, Product Nr. r119.aq). Reverse phase chromatography was performed at 50 °C with an EASY-nLC 1000 ultra-high-pressure system (Thermo Fisher Scientific) coupled to the Q Exactive mass spectrometer (Thermo Fisher Scientific) via a nano-electrospray source (Thermo Fisher Scientific). Peptides were separated by a linear gradient of buffer B up to 40% in 240 min for a 4-h gradient run with a flow rate of 250 nl/min. The Q Exactive was operated in the data-dependent mode with survey scans (MS resolution: 50,000 at m/z 400) followed by up to the top 10 MS2 method selecting ≥2 charges from the survey scan with an isolation window of 1.6 Th and fragmented by higher energy collisional dissociation with normalized collision energies of 25. Repeated sequencing was avoided using a dynamic exclusion list of the sequenced precursor masses for 40 s.
6
Peptide Separation and Identification by LC-MS
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Peptides were separated with a Waters NanoAcquity UPLC and emitted into a Thermo Q-Exactive HF tandem mass spectrometer. Pulled tip columns were created from 75 μm inner diameter fused silica capillary in-house using a laser pulling device and packed with 2.1 μm C18 beads (Dr. Maisch GmbH) to 300 mm. Trap columns were created from 150 μm inner diameter fused silica capillary fritted with Kasil on one end and packed with the same C18 beads to 25 mm. Buffer A was water and 0.1% formic acid, while buffer B was 98% acetonitrile and 0.1% formic acid. For each injection, 3 μl of each sample was loaded with 5 μL 2% B and eluted using the following program: 0–90 minutes
2%-35% B, 90–100 minutes 35%–60% B, followed by a 35 min washing gradient. Data were acquired using data-dependent acquisition (DDA).
2%-35% B, 90–100 minutes 35%–60% B, followed by a 35 min washing gradient. Data were acquired using data-dependent acquisition (DDA).
7
SILAC-based Proteomics Workflow
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Protein extracts from light and heavy N2a cells were obtained by homogenization in lysis buffer containing 8 M urea in 50 mM ABC (pH 8.2) supplemented with complete protease and phosphatase inhibitor cocktails (Roche), followed by sonication (three 10 s pulses with 30 s on ice between each pulse). Protein concentrations were determined using the Bradford assay (Bio-Rad). Lysates from light and heavy N2a cells were mixed at a 1:1 ratio and loaded onto 30-kDa molecular weight cutoff Microcon filters (Millipore; Billerica, MA, USA). Proteins were reduced by incubating samples with 20 mM DTT for 30 min at 37°C with agitation (245 rpm) and subsequently alkylated with 20 mM IAA for 30 min in darkness at room temperature. Protein digestion was performed by incubation with 40:1 (w/w, protein:enzyme) trypsin (Worthington Biochemical Corporation) overnight at 37°C with agitation (245 rpm). Samples were acidified using 10% (v/v) FA, then desalted using inhouse made C18 desalting cartridges (C18 beads: Dr. Maisch GmbH) and desiccated using a SpeedVac prior to being resuspended in 0.1% (v/v) FA for LC-MS/MS analysis.
8
SILAC Peptide Mixtures Identification
9
Nanocolumn-based Peptide Separation and MS Analysis
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Samples were injected on a 15 cm nanocolumn (75 μm inner diameter) packed with 1.9 μm C18 beads (Dr. Maisch GmbH, Entringen, Germany) using an Easy-LC 1200 (Thermo Fisher Scientific). Peptides were separated and eluted from the column with an increasing gradient of buffer B (80% acetonitrile, 0.1% formic acid) at a flow rate of 250 nL/minute.
All samples were analyzed on a Q-Exactive HF-X (Thermo Fisher Scientific) mass spectrometer coupled to EASY-nLC 1200. Except for two replicates of in-gel TTP pulldown and one replicate from PAC TTP pulldown experiments were analyzed on a Lumos (Thermo Fisher Scientific) mass spectrometer with similar scan settings. The mass spectrometer was operated in positive mode with TopN method.
All samples were analyzed on a Q-Exactive HF-X (Thermo Fisher Scientific) mass spectrometer coupled to EASY-nLC 1200. Except for two replicates of in-gel TTP pulldown and one replicate from PAC TTP pulldown experiments were analyzed on a Lumos (Thermo Fisher Scientific) mass spectrometer with similar scan settings. The mass spectrometer was operated in positive mode with TopN method.
10
Metabolomic Analysis of Yeast Samples
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The reagents included the following: LC-MS grade acetonitrile (ACN); water; ammonium acetate; ammonium sulfate; ammonium hydroxide; and formic acid (Sigma-Aldrich, St. Louis, MD, USA); 0.5 mm disruption beads (RPI); DifcoTM yeast nitrogen base (BD Bioscience, San Jose, CA, USA); dextrose (Fisher Bioreagents, Fair Lawn, NJ, USA); 13C-6 glucose; 15N-2 ammonium sulfate (Cambridge Isotope Laboratories, Tewksbury, MA, USA); Mass Spectrometry Metabolite Library of Standards (IROA Technologies, Sea Girt, NJ, USA); 1.9 µm C18 beads (Dr. Maisch GmbH, Germany); 75 µm ID empty silica column (New Objective, Woburn, MA, USA); and SeQuant ZIC-HILIC column (MilliporeSigma, Burlington, VT, USA).
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