20 cm fused silica emitter

Manufactured by New Objective

Sourced in Germany

The 20 cm fused silica emitter is a laboratory equipment item designed for specific technical applications. It is made of fused silica, a type of glass known for its high purity and resistance to thermal and chemical stresses. The emitter has a length of 20 centimeters. No further details about its intended use or function can be provided in an unbiased and factual manner.

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Fused-silica emitter (6 citations)

5 protocols using 20 cm fused silica emitter

Quantitative Proteome Analysis by LC-MS/MS

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Proteins were separated on 4–12% gradient NuPAGE Novex Bis-Tris gel (Life Technologies) and visualized using Instant blue (Expedeon). Each gel lane was excised into six slices, which were reduced using 10 mM dithiothreitol, alkylated with 55 mM iodoacetamide, and digested with trypsin (Trypsin gold, Promega), overnight at 35 °C. Tryptic peptides were desalted and dried in a centrifugal evaporator. Dried tryptic peptides were resuspended in and loaded with a buffer containing 2% acetonitrile and 0.1% formic acid, and separated using a 20 cm fused silica emitter (New Objective) packed in-house with reverse-phase Reprosil-Pur Basic 1.9 μm (Dr. Maisch GmbH). Tryptic peptides were analysed on an Orbitrap Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled online to an EASY-nLC II (Thermo Fisher Scientific). For the full scan a resolution of 60,000 at 250 Th was used. The top ten most intense ions in the full MS were isolated for fragmentation with a target of 50,000 ions at a resolution of 15,000 at 250 Th. MS data were acquired using the XCalibur software (Thermo Fisher Scientific - Version 4.2.28.12).

Marco S., Neilson M., Moore M., Perez-Garcia A., Hall H., Mitchell L., Lilla S., Blanco G.R., Hedley A., Zanivan S, & Norman J.C. (2021). Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion. Nature Communications, 12, 6829.

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Peptide Separation and Analysis

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Tryptic peptides were separated on 20 cm fused silica emitter (New Objective, Woburn, MA) packed in-house with the reverse phase ReproSil-Pur C₁₈-AQ, 1.9 μm resin (Dr. Maisch, GmbH, Ammerbuch-Entringen, Germany) and analyzed on a LTQ-Orbitrap Elite (Thermo Fisher Scientific) coupled on-line with a nano-HPLC (Easy nLC, Thermo Fisher Scientific).

Patella F., Schug Z.T., Persi E., Neilson L.J., Erami Z., Avanzato D., Maione F., Hernandez-Fernaud J.R., Mackay G., Zheng L., Reid S., Frezza C., Giraudo E., Fiorio Pla A., Anderson K., Ruppin E., Gottlieb E, & Zanivan S. (2015). Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability. Molecular & Cellular Proteomics : MCP, 14(3), 621-634.

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Nanoscale Liquid Chromatography Mass Spectrometry

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Tryptic peptides were separated by nanoscale C18 reverse-phase liquid chromatography using an EASY-nLC 1200 (Thermo Fisher Scientific) coupled online to an Orbitrap Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) via nanoelectrospray ion source (Thermo Fisher scientific). Peptides were separated over a 60-minute gradient on a 20-cm fused silica emitter (New Objective) packed in house with reverse-phase Reprosil Pur Basic 1.9 μm (Dr. Maisch GmbH). For the full scan, a resolution of 60,000 at 250 Th was used. The top 10 most intense ions in the full MS were isolated for fragmentation with a target of 50,000 ions at a resolution of 15,000 at 250 Th. MS data were acquired using the XCalibur software (Thermo Fisher Scientific).

Singh S.P., Thomason P.A., Lilla S., Schaks M., Tang Q., Goode B.L., Machesky L.M., Rottner K, & Insall R.H. (2020). Cell–substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime. PLoS Biology, 18(8), e3000774.

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Nanoscale Peptide Separation and MS Analysis

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Each of the 21 fractions was then re-suspended in 2% acetonitrile/0.1% TFA acid in water and separated by nanoscale C18 reverse-phase liquid chromatography performed on an EASY-nLC II (Thermo Scientific) coupled to a Linear Trap Quadrupole - Orbitrap Velos mass spectrometer (Thermo Scientific). Elution was carried out using a binary gradient with buffer A: 2% acetonitrile and B: 80% acetonitrile, both containing 0.1% of formic acid. Peptides were subsequently eluted at 200 nl/min flow, into a 20 cm fused silica emitter (New Objective) packed in-house with ReproSil-Pur C18-AQ, 1.9 μm resin (Dr Maisch GmbH). Packed emitter was kept at 35°C by means of a column oven integrated into the nanoelectrospray ion source (Sonation). Peptides were eluted at a flow rate of 200 nl/min using 3 different gradients optimised for set of fractions 1-7 (2,20,41% buffer B), 8-15 (5, 25, 46% buffer B) and 16-21 (7, 28, 50% buffer B). Two-step gradients were used, all with 42 minutes for step one and 13 minutes for step two.All gradients were followed by a washing step (100% B) for 10 minutes followed by a 20 minute re-equilibration step (5%), for a total run time of 85 minutes.
Eluting peptides were electrosprayed into the mass spectrometer using a nanoelectrospray ion source (Thermo Scientific). An Active Background Ion Reduction Device was used to decrease air contaminants signal level.

Salji M.J., Blomme A., Däbritz J.H., Repiscak P., Lilla S., Patel R., Sumpton D., van den Broek N.J., Daly R., Zanivan S, & Leung H.Y. (2022). Multi-omics & pathway analysis identify potential roles for tumor N-acetyl aspartate accumulation in murine models of castration-resistant prostate cancer. iScience, 25(4), 104056.

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Orbitrap-based Shotgun Proteomics

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Digested peptides were injected on an EASY‐nLC system coupled on line to a LTQ‐Orbitrap Elite via a nanoelectrospray ion source (Thermo Scientific). Peptides were separated using a 20‐cm fused silica emitter (New Objective) packed in house with reversed‐phase Reprosil Pur Basic 1.9 μm (Dr. Maisch GmbH). MS data were acquired using the Xcalibur software (Thermo Scientific) and .raw files processed with the MaxQuant computational platform (Cox & Mann, 2008) and searched with the Andromeda search engine (Cox et al, 2011) against the human UniProt Consortium (2010) database (release‐2012 01, 88,847 entries). See also Appendix Supplementary Methods.

Reid S.E., Kay E.J., Neilson L.J., Henze A., Serneels J., McGhee E.J., Dhayade S., Nixon C., Mackey J.B., Santi A., Swaminathan K., Athineos D., Papalazarou V., Patella F., Román‐Fernández Á., ElMaghloob Y., Hernandez‐Fernaud J.R., Adams R.H., Ismail S., Bryant D.M., Salmeron‐Sanchez M., Machesky L.M., Carlin L.M., Blyth K., Mazzone M, & Zanivan S. (2017). Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium. The EMBO Journal, 36(16), 2373-2389.

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