Easy spray pepmap c18 column

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Easy Spray PepMap C18 column is a reversed-phase HPLC column designed for the separation of peptides and proteins. It features a porous silica-based stationary phase with a C18 functional group, which provides high-resolution separation of complex samples. The column is suitable for a range of applications, including proteomics, metabolomics, and pharmaceutical analysis.

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Easy Spray PepMap C18 analytical column EASY-Spray PepMap RSLC C18 column EASY-Spray Acclaim PepMap C18 Column EASY-Spray PepMap C18 75 μm × 250 mm column PepMap C18 EASY-Spray LC column EASY-Spray PepMap RSLC C18 75 µm × 250 mm column EASY-Spray PepMap RSLC C18 75μm × 250mm column ES803 Easy-Spray PepMap C18 Column EASY-Spray Pepmap C18 trap column Easy-Spray C18 PepMap

26 protocols using easy spray pepmap c18 column

Proteomics and Serine Incorporation Analysis

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Proteomics analysis was performed as described previously [57] . Briefly, yeast cells were lysed using the filter aided sample preparation method [58] (link). For full proteome analysis, samples were eluted from a PepMap C18 easy spray column (Thermo) with a linear gradient of acetonitrile from 10-35% in H2O with 0.1% formic acid for 118 min at a constant flow rate of 300 nl/min.
For serine incorporation assays using [ 13 C3 15 N1]-serine (Cambridge Isotope Labs) samples were eluted from a PepMap C18 easy spray column (Thermo) with a linear gradient of acetonitrile from 10-50% in 0.1% formic acid for 33 min at a constant flow rate of 300 nl/min.
The resulting MS and MS/MS spectra were analyzed using MaxQuant (version 1.6.0.13, www. maxquant.org/; Cox et al., 2011; (link)Cox and Mann, 2008) (link) as described previously [57] . For incorporation tests, [ 13 C3 15 N1]-serine was added as a modification to the MaxQuant database (mass change =4.0070994066 Da). For the calculation of incorporation rates, the peptide list was filtered for serine containing peptides with a valid heavy/light ratio. For each peptide, the incorporation was calculated as 1 -(1/(ratio H/L -1)). The maximum of a density distribution of all peptides represents the estimated incorporation level. All calculations and plots were performed with the R software package (www.r-project.org/; RRID:SCR_001905).

Esch B.M., Limar S., Bogdanowski A., Gournas C., More T.H., Sundag C., Walter S., Heinisch J.J., Ejsing C.S., André B., & Fröhlich F. (2020). Uptake of exogenous serine is important to maintain sphingolipid homeostasis inSaccharomyces cerevisiae.

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Mass Spectrometry-Based Proteomics Workflow

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Each sample was analyzed by LC/MS/MS using an Easy nLC 1000 coupled to a QExactive HF (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Scientific) and separated over a 60 min method. The gradient for separation consisted of 5 – 32% mobile phase B at a 250 nL/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent HCD fragmentation. Resolution for the precursor scan (m/z 350 – 1600) was set to 120,000, while MS/MS scan resolution was set to 15,000. The normalized collision energy was set to 27% for HCD. Peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥ 7 were excluded.

O’Banion C.P., Priestman M.A., Hughes R.M., Herring L.E., Capuzzi S.J, & Lawrence D.S. (2017). Design and Profiling of a Subcellular Targeted Optogenetic cAMP-Dependent Protein Kinase. Cell chemical biology, 25(1), 100-109.e8.

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Nano-UHPLC and Orbitrap Mass Spectrometry

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For mass spectrometry analysis, peptides were separated at a flow rate of 300 nl/min on a 50 cm long, 75 µm internal diameter EASY-spray PepMap C18 column (Thermo Fisher Scientific) using a Dionex UltiMate 3000 Nano-UHPLC system (Thermo Fisher Scientific). The column was maintained at 50 °C. Buffers A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted in 165 min runs, with a gradient from 3% to 5% buffer B for 5 min, from 5% to 25% buffer B for 105 min, from 25% to 35% buffer B for 15 min, from 35% to 95% buffer B for 20 min, and equilibration (3% buffer B) for 19 min. Eluting peptides were analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). The instrument was operated in a data-dependent, “Top Speed” mode, with cycle times of 3 s. Peptide precursor mass to charge ratio (m/z) measurements (MS1) were carried out at 120,000 resolution in the 375–1500m/z range. The MS1 AGC target was set to 4E^{5 (link)} and the maximum injection time to 50 ms. Precursor priority was set to “most intense” and precursors with charge states 2–7 only were selected for HCD fragmentation. Fragmentation was carried out using 35% collision energy. The m/z of the peptide fragments were measured in the ion trap using an AGC target of 1E⁴ and 50 ms maximum injection time.

Ng P.Y., Ribet A.B., Guo Q., Mullin B.H., Tan J.W., Landao-Bassonga E., Stephens S., Chen K., Yuan J., Abudulai L., Bollen M., Nguyen E.T., Kular J., Papadimitriou J.M., Søe K., Teasdale R.D., Xu J., Parton R.G., Takayanagi H, & Pavlos N.J. (2023). Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts. Nature Communications, 14, 906.

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Quantitative Proteomics of Biological Samples

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Each sample was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1200 coupled to a QExactive HF (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Scientific) and separated over a 120 min method. The gradient for separation consisted of a step gradient from 5 to 35 to 45% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile (ACN). The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent HCD fragmentation. Resolution for the precursor scan (m/z 300–1600) was set to 120,000 with a target value of 3 × 10⁶ ions, 100 ms inject time. MS/MS scans resolution was set to 15,000 with a target value of 1 × 10⁵ ions, 75 ms inject time. The normalized collision energy was set to 27% for higher-energy C-trap dissociation (HCD), with an isolation window of 1.6 m/z. Peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded. All peptides identified by LC/MS/MS are listed in Table S1.

Evangelista B.A., Cahalan S.R., Ragusa J.V., Mordant A., Necarsulmer J.C., Perna R.J., Ajit T., White K., Barker N.K., Tian X., Cohen S., Meeker R., Herring L.E, & Cohen T.J. (2023). Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates. iScience, 26(5), 106645.

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Affinity Purification-MS Protocol for Peptides

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Peptide samples from both the MIB-MS and AP-MS sample sets were analyzed by LC-MS/MS using an Easy nLC 1200 coupled to a QExactive HF mass spectrometer (Thermo Fisher Scientific). Samples were injected onto an EASY-Spray PepMap C18 column (75 μm inner diameter × 25 cm, 2 μm particle size; Thermo Fisher Scientific) and separated over a 120-min method. The gradient for separation consisted of 5 to 40% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in 80% acetonitrile. The QExactive HF was operated in data-dependent mode, where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (mass/charge ratio, 350 to 1600) was set to 120,000 with a target value of 3 × 10⁶ ions. MS/MS scan resolution was set to 15,000 with a target value of 5 × 10⁴ ions, 60 ms maximum injection time. The normalized collision energy was set to 27% for high collision dissociation. Dynamic exclusion was set to 30 s, peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥ 7 were excluded.

Wass A.B., Krishna B.A., Herring L.E., Gilbert T.S., Nukui M., Groves I.J., Dooley A.L., Kulp K.H., Matthews S.M., Rotroff D.M., Graves L.M, & O’Connor C.M. (2022). Cytomegalovirus US28 regulates cellular EphA2 to maintain viral latency. Science Advances, 8(43), eadd1168.

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Peptide Analysis by LC/MS/MS

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The peptide samples (n=3) were analyzed by LC/MS/MS using an Easy nLC 1000 coupled to a QExactive HF mass spectrometer (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size; Thermo Scientific) and separated over a 2 hr method. The gradient for separation consisted of 5–32% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in ACN. The QExactive HF was operated in datadependent mode where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (m/z 400–1600) was set to 120,000 with a target value of 3 × 10⁶ ions. MS/MS scans resolution was set to 15,000 with a target value of 2 × 10⁴ ions. The normalized collision energy was set to 27% for HCD. Peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded.

Vaseva A.V., Blake D.R., Gilbert T.S., Ng S., Hostetter G., Azam S.H., Ozkan-Dagliyan I., Gautam P., Bryant K.L., Pearce K.H., Herring L.E., Han H., Graves L.M., Witkiewicz A.K., Knudsen E.S., Pecot C.V., Rashid N., Houghton P.J., Wennerberg K., Cox A.D, & Der C.J. (2018). KRAS Suppression-Induced Degradation of MYC is Antagonized by a MEK5-ERK5 Compensatory Mechanism. Cancer cell, 34(5), 807-822.e7.

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Quantitative Proteomics by LC-MS/MS

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Samples were normalized and 0.6 μg of each sample was analyzed by LC-MS/MS using an Easy nLC 1200 coupled to a Fusion Lumos (Thermo Fisher Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Fisher Scientific) and separated over a 120 min method. The gradient for separation consisted of 5-32-45% mobile phase B at a 250 nL/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% acetonitrile. Fusion Lumos was operated in data-dependent mode with a 3 s cycle time. Resolution for the precursor scan (m/z 350–1500) was set to 120,000 with a 250% AGC target, 50 ms inject time. MS/MS scans acquired in the Orbitrap with a resolution set to 15,000, 250% AGC target, 50 ms inject time. The normalized collision energy was set to 30% for higher energy collisional dissociation (HCD), with an isolation window of 1.6 m/z. MIPS was on, and precursors with unknown charge or a charge state of 1 and ≥ 7 were excluded.

Gentile G.M., Gamarra J.R., Engels N.M., Blue R.E., Hoerr I., Wiedner H.J., Hinkle E.R., Cote J.L., Leverence E., Mills C.A., Herring L.E., Tan X, & Giudice J. (2022). The synaptosome-associated protein-23 (SNAP23) is necessary for proper myogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(8), e22441.

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Quantitative Proteome Analysis via SPS-MS3

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Twenty-four proteome fractions were analyzed by LC–MS/MS using an Easy nLC 1200 coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Scientific) and separated over a 120-min method. The gradient for separation consisted of 5–42% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN.
For the proteome fractions, the Lumos was operated in SPS-MS3 mode [119 (link)], with a 3-s cycle time. Resolution for the precursor scan (m/z 350–2000) was set to 120,000 with a AGC target set to standard and a maximum injection time of 50 ms. MS2 scans consisted of CID normalized collision energy (NCE) 30; AGC target set to standard; maximum injection time of 50 ms; isolation window of 0.7 Da. Following MS2 acquisition, MS3 spectra were collected in SPS mode (10 scans per outcome); HCD set to 65; resolution set to 50,000; scan range set to 100–500; AGC target set to 200% with a 150 ms maximum inject time.

Garcia E.L., Steiner R.E., Raimer A.C., Herring L.E., Matera A.G, & Spring A.M. (2024). Dysregulation of innate immune signaling in animal models of spinal muscular atrophy. BMC Biology, 22, 94.

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Peptide Analysis by LC-MS/MS

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The peptide samples were analyzed by LC/MS/MS using an Easy nLC 1200 coupled to a QExactive HF Biopharma mass spectrometer (Thermo Fisher Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id×25 cm, 2 μm particle size) (Thermo Fisher Scientific) and separated over a 2 h method. The gradient for separation consisted of 5-45% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile (ACN). The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (m/z 350-1700) was set to 60,000, while MS/MS scans resolution was set to 15,000. The normalized collision energy was set to 27% for HCD. Peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥7 were excluded.

Cherney R.E., Mills C.A., Herring L.E., Braceros A.K, & Calabrese J.M. (2023). A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB. Biology Open, 12(6), bio059955.

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Glycopeptide Enrichment and Analysis

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Enriched glycopeptides from BSM were trapped on a C18 Acclaim PepMap RSLC column (3.0 μm, 75 μm × 20 mm; Thermo Fisher Scientific) using a loading pump solvent composed of 2% acetonitrile and 0.05% formic acid in water. The trapped glycopeptides were separated on an EASY-Spray PepMap C18 column (2.0 μm, 75 μm × 500 mm; Thermo Fisher Scientific) using NC pump solvents for glycopeptide analysis. NC pump solvents consisted of mobile phase A (0.1% formic acid in aqueous solution) and mobile phase B (0.1% formic acid in acetonitrile). Samples were separated with a 2%–40% linear gradient of solvent B over 50 min at a flow rate of 200 nL/min. The mass spectrometry (MS) parameters used were the same as previously reported [19 (link)]. The spectral interpretation was carried out according to the procedure of Segu et al. [20 (link)]. Briefly, Y₁ ions of the glycopeptides were indicated by distinct peaks as the most abundant ion in the MS/MS spectrum, and the other peaks were determined as their composition through the different mass values based on Y₁.

Kim J., Ryu C., Ha J., Lee J., Kim D., Ji M., Park C.S., Lee J., Kim D.K, & Kim H.H. (2020). Structural and Quantitative Characterization of Mucin-Type O-Glycans and the Identification of O-Glycosylation Sites in Bovine Submaxillary Mucin. Biomolecules, 10(4), 636.

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