Pepmap rslc c18 easyspray column

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PepMap RSLC C18 EasySpray column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of peptides. It features a C18 stationary phase and is specifically optimized for use in nano-HPLC and capillary HPLC systems.

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11 protocols using pepmap rslc c18 easyspray column

Synthetic Peptide MS Analysis

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Peptide mixtures were dissolved in loading buffer (1% Acetonitrile, 0.1% Trifluoroacetic acid), and 200 fmols/peptide were analyzed by an Ultimate 3000 HPLC system coupled to a high field Q-Exactive (HFX) Orbitrap mass spectrometer (Thermo Scientific). Peptides were trapped by PepMap 100 C18 columns (ThermoFisher Scientific) before reverse phase separation with a 60 min gradient of acetonitrile 2% to 25%, in 1% DMSO, 0.1% Formic acid at a flow rate of 250 nl/min on a 75 μm × 50 cm PepMap RSLC C18 EasySpray column (ThermoFisher Scientific). Data-dependent acquisition involved one full MS1 scan (120,000 resolution, 60 ms accumulation time, AGC 3 × 10⁶) followed by 20 data-dependent MS2 scans (60,000 resolution, 120 ms accumulation time, AGC 5 × 10⁵), with an isolation width of 1.6 m/z and normalized HCD energy of 25%. Three methods were utilized for analysis of the synthetic standard: (A) considered charge states of 2 to 4, (B) considered charge states of 1 to 4 while (C) involved one full scan 300 to 700 followed by 18 MS2 scans for charge states 2 to 4 followed by one full scan 700 to 1400 followed by two MS2 scans for charge states 1. Dynamic exclusion was set for 30 s. For enzymatic digests normalized HCD was increased to 28% and only 2 to 4 charge states were acquired.

Parker R., Tailor A., Peng X., Nicastri A., Zerweck J., Reimer U., Wenschuh H., Schnatbaum K, & Ternette N. (2021). The Choice of Search Engine Affects Sequencing Depth and HLA Class I Allele-Specific Peptide Repertoires. Molecular & Cellular Proteomics : MCP, 20, 100124.

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Proteomic Profiling using Orbitrap LC-MS/MS

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Aliquots (each containing around 3 μg of digested material) of 11 non-consecutive chromatographic fractions were run onto a 2 μm × 75 μm × 50 cm PepMap RSLC C18 EasySpray column (Thermo Scientific) using 3-h MeCN gradients (2–30% in 0.1% formic acid), and were used to elute peptides at a flow rate of 300 nl min⁻¹ for analysis in an Orbitrap Lumos Fusion mass spectrometer (Thermo Scientific) in positive ion mode. MS spectra were acquired at 375–1,500 m/z with a resolution of 120,000. For each MS spectrum, multiply charged ions above the selected threshold (2 × 10⁴) were selected for tandem MS (MS–MS) in cycles of 3 s with an isolation window of 0.7 m/z. Precursor ions were fragmented by HCD using stepped relative collision energies of 30, 35 and 40 to ensure efficient generation of both sequence ions and TMT reporter ions. MS–MS spectra were acquired in centroid mode with resolution 50,000 and 110 m/z. A dynamic exclusion window was applied, which prevented selection of the same level of m/z for 30 s after acquisition.

Grossman Z, & Paul W.E. (1992). Adaptive cellular interactions in the immune system: the tunable activation threshold and the significance of subthreshold responses.. Proceedings of the National Academy of Sciences of the United States of America, 89(21), 10365-10369.

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Proteomic Analysis by Mass Spectrometry

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Mass spectrometry analyses were performed on a Q-Exactive Plus hybrid quadripole-orbitrap mass spectrometer (Thermo Fisher Scientific, San José, CA, USA) coupled to an Easy 1000 reverse phase nano-flow LC system (Proxeon) using the Easy nano-electrospray ion source (Thermo Fisher Scientific). Each peptide mixture was analyzed in duplicate. Five μL were loaded onto an Acclaim PepMap precolumn (75 μm × 2 cm, 3 μm, 100 Å; Thermo Fisher Scientific) equilibrated in solvent A and separated at a constant flow rate of 250 nL/min on a PepMap RSLC C18 Easy-Spray column (75 μm × 50 cm, 2 μm, 100 Å; Thermo Fisher Scientific) with a 90-min gradient (0% to 20% B solvent [0.1% (vol/vol) formic acid in acetonitrile] in 70 min and 20% to 37% B solvent in 20 min).
Data acquisition was performed in positive and data-dependent modes. Full scan MS spectra (mass range m/z 400 to 1,800) were acquired in centroïd with a resolution of 70,000 (at m/z 200) and MS/MS spectra were acquired in centroid mode at a resolution of 17,500 (at m/z 200). All other parameters were kept as described in (30 (link))

Allouche D., Kostova G., Hamon M., Marchand C.H., Caron M., Belhocine S., Christol N., Charteau V., Condon C, & Durand S. (2023). New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC. Microbiology Spectrum, 11(4), e00471-23.

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Proteomics of Chromatographic Fractions

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Aliquots (containing around 5 μg of digested material) of 10 nonconsecutive chromatographic fractions were run onto a 2-μm, 75 μm × 50 cm PepMap RSLC C18 EasySpray column (Thermo Fisher Scientific) using 3-hour acetonitrile gradients (2 to 25% in 0.1% formic acid) to elute peptides, at a flow rate of 200 nl/min, for analysis in a QExactive Plus Orbitrap MS (Thermo Fisher Scientific) in positive ion mode. MS spectra were acquired between 350 and 1500 mass/charge ratio (m/z) with a resolution of 70,000. For each MS spectrum, the 10 most intense multiply charged ions over the selected threshold (1.7 × 10⁴) were selected for tandem MS (MS/MS) with an isolation window of 1 m/z. Precursor ions were fragmented by HCD using stepped relative collision energies of 25, 35, and 40 to ensure efficient generation of sequence ions and TMT reporter ions. MS/MS spectra were acquired in centroid mode with a resolution of 70,000 from m/z = 100. A dynamic exclusion window was applied that prevented the same m/z from being selected for 10 s after its acquisition.

Forester C.M., Oses-Prieto J.A., Phillips N.J., Miglani S., Pang X., Byeon G.W., DeMarco R., Burlingame A., Barna M, & Ruggero D. (2022). Regulation of eIF4E guides a unique translational program to control erythroid maturation. Science Advances, 8(51), eadd3942.

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Proteomic Analysis of Mouse Corona

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The resulting samples were further analyzed by electrospray liquid chromatography–tandem mass spectrometry using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled online to an EASY-nLC 1200 (Thermo Fisher Scientific). Samples were run at a flow rate of 300 nL/min using the following gradient: 0 to 4 min 0 to 5% B, 4 to 74 min 0 to 30% B, 74 to 79 min 30 to 90% B, 79 to 89 min 90% B, 89 to 94 min at 90 to 0% B, and 94 to 105 min 0% B for column re-equilibration. Mobile phase A was 96.1:3.9 0.1% formic acid (FA) in water/0.1% FA in acetonitrile (ACN). Mobile phase B was 80.0:20.0 0.1% FA in water/0.1% FA in ACN. The samples were first desalted on a Thermo Fisher Scientific Acclaim PepMap 100 C18 HPLC column (3 μm particle size, 75 μm × 2 cm, 100 Å) before separation on a Thermo Fisher Scientific PepMap RSLC C18 EASY-Spray Column (3 μm particle size, 75 μm × 15 cm, 100 Å). The data were analyzed using the Proteome Discoverer 2.1.0.81 software (Thermo Fisher Scientific). The data were run against the Mus musculus Uniprot FASTA file (modified 26 August 2020) to identify the mouse proteins in the corona.

Qiu M., Tang Y., Chen J., Muriph R., Ye Z., Huang C., Evans J., Henske E.P, & Xu Q. (2022). Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis. Proceedings of the National Academy of Sciences of the United States of America, 119(8), e2116271119.

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Peptide Separation and Mass Spectrometry

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Each fraction of enriched peptides was run in technical replicate, injecting approximately half each time. Peptides were separated on an Ultimate 3000 HPLC with loading on a 5 cm C18 trap column Acclaim™ PepMap™ 100 (3 µm particle size, 75 µm diameter; Thermo Scientific, Cat No: 164946) followed by a 15 cm PepMap RSLC C18 EASY-Spray column (Thermo Scientific, Cat No: ES900) and analyzed using a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) operated in positive ion mode. After loading the trap column at 3 µL/min for 7 min, the column was switched inline and at 400 nL/min peptides were separated 5–35% solvent B over 60 min, 8–50% solvent B over 30 min, 50–95% solvent B over 1 min, held at 95%B for 2 min and 95–8% B over 1 min (Solvent A: 95% water, 5% acetonitrile, 0.1% formic acid; Solvent B: 100% acetonitrile, 0.1% formic acid). A data-dependent TopN20 mass acquisition method was used with the following parameters for the MS1: mass resolution: 70,000; AGC target: 3e6; maximum IT: 100 ms; and mass scan range: 300–2000 m/z. MS2 parameters were fixed first mass: 100 m/z; resolution: 17,500; AGC target: 1e5; maximum IT: 50 ms; isolation window: 4.0 m/z; normalized collision energy: 30; dynamic exclusion: 30 s; and charge exclusion: 1, 7, 8, > 8.

Berryhill C.A., Hanquier J.N., Doud E.H., Cordeiro-Spinetti E., Dickson B.M., Rothbart S.B., Mosley A.L, & Cornett E.M. (2023). Global lysine methylome profiling using systematically characterized affinity reagents. Scientific Reports, 13, 377.

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Quantitative Proteome Analysis by LC-MS

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For the 2 conditions (i.e. ± 1 mM GSNO), 4 biological replicates were analyzed as technical triplicates on a Q Exactive Plus hybrid quadripole-orbitrap mass spectrometer (Thermo Fisher, San José, CA, USA) coupled to an Easy 1000 reverse phase nano-flow LC system (Proxeon) using the Easy nano-electrospray ion source (Thermo Fisher). Five microliters of peptide mixtures were injected onto an Acclaim PepMap precolumn (75 µm × 2 cm, 3 µm, 100 Å; Thermo Scientific) equilibrated in buffer A and separated at a constant flow rate of 250 nL/min on a PepMap RSLC C18 EASY-Spray column (75 µm × 50 cm, 2 µm, 100 Å; Thermo Scientific) with a 90 min gradient (0% to 20% B solvent (0.1% [v/v] formic acid in acetonitrile) in 70 min and 20% to 32% B solvent in 20 min). Data were acquired in Data Dependent mode as described in Pérez-Pérez et al. (2017).

Lambert L., de Carpentier F., André P., Marchand C.H, & Danon A. (2023). Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii. Plant Physiology, 194(4), 2648-2662.

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Liquid Chromatography-based Quantitative Proteomics

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Liquid chromatography was performed on an EASY-nLC1200 liquid chromatography system (Thermo Fisher Scientific) in single-pump trapping mode with a PepMap RSLC C18 EASY-spray column (2 μm, 100 Å, 75 μm × 25 cm) and a Pepmap C18 trap column (3 μm, 100 Å, 75 μm × 20 mm). Samples were separated at 300 nL/min with a 260 min linear gradient (3–85% solvent containing 80% acetonitrile with 0.1% formic acid), and data were acquired on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) in data-dependent mode. A full scan was conducted using 60k resolution in the Orbitrap in positive mode. Precursors for mass spectrometry 2 (MS2) were filtered by monoisotopic peak determination for peptides. Intensity threshold was set to 5.0 × 10³, charge states 2–7 were selected, and dynamic exclusion was set to 60 s after one analysis with a mass tolerance of 10 ppm. Collisionally induced dissociation spectra were collected in ion trap MS² at 35% energy and isolation window 1.6 m/z. Synchronous precursor selection MS³ was utilized for TMT ratio determination at higher-energy C-trap dissociation (HCD) energy. The sequence of steps for TMT analysis are shown in Figure 1.

Lee C.H., Tang J.C., Hendricks N.G, & Anvari B. (2023). Proteomes of Micro- and Nanosized Carriers Engineered from Red Blood Cells. Journal of proteome research, 22(3), 896-907.

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Proteolytic Enzyme Digestion and LC-MS/MS Analysis

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Samples consisted of approximately 1 μM of in-solution digested product. For each proteolytic, enzyme digestion liquid chromatography was performed on a Thermo nLC1200 (Thermo Fisher Scientific Inc., Waltham, MA, USA) in single-pump trapping mode with a Thermo PepMap RSLC C18 EASY-spray column (2 μm, 100 Å, 75 μm × 25 cm) and a Pepmap C18 trap column (3 μm, 100 Å, 75 μm × 20 mm). The solvents used were A: water with 0.1% formic acid and B: 80% acetonitrile with 0.1% formic acid. Samples were separated at 300 nL/min with a 250 min gradient starting at 3% B increasing to 30% B from 1 to 231 min, then to 85% B at 241 min, holding for 10 min.
Mass spectrometry data were acquired on a Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in data-dependent mode. A full scan was conducted using 60 k resolution in the Orbitrap in positive mode. Precursors for MS² were filtered by monoisotopic peak determination for peptides, intensity threshold 5.0 × 10³, charge state 2–7, and 60 s dynamic exclusion after 1 analysis with a mass tolerance of 10 ppm. Higher-energy C-trap dissociation (HCD) spectra were collected in ion trap MS² at 35% energy and isolation window 1.6 m/z.

Madahar V., Dang R., Zhang Q., Liu C., Rodgers V.G, & Liao J. (2023). Human Post-Translational SUMOylation Modification of SARS-CoV-2 Nucleocapsid Protein Enhances Its Interaction Affinity with Itself and Plays a Critical Role in Its Nuclear Translocation. Viruses, 15(7), 1600.

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Liquid Chromatography-Mass Spectrometry Protocol

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Thermo nLC1200, in single-pump trapping mode, was used with a Thermo PepMap RSLC C18 EASY-spray column (2 μm, 100 Å, 75 μm x 25 cm) and a PepMap C18 trap column (3 μm, 100 Å, 75 μm x 20 cm) to perform liquid chromatography. Solvent A was water with 0.1% formic acid and solvent B was 80% acetonitrile with 0.1% formic acid. Samples were separated at 300 nL/min with a 250-minute gradient starting at 3% B, increasing to 30% B from 1–231 minutes, and then 85% B at 241 minutes, holding for 10 minutes.
Mass spectrometry was performed on a Thermo Orbitrap Fusion in a data-dependent mode. A full scan was conducted using 60k resolution in the Orbitrap in positive mode. Precursors for MS/MS were filtered by monoisotopic peak determination for peptides, intensity threshold 5.0e3, charge state 2–7, and 60 second dynamic exclusion after 1 analysis with a mass tolerance of 10 ppm. Higher energy C-trap dissociation (HCD) spectra were collected in ion trap MS/MS at 35% energy and isolation window 1.6 m/z.

Coreas R., Cao X., Deloid G.M., Demokritou P, & Zhong W. (2020). Lipid and protein corona of food-grade TiO2 nanoparticles in simulated gastrointestinal digestion. NanoImpact, 20, 100272.

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