Reprosil c18 resin

Manufactured by Dr. Maisch

Sourced in Germany

Reprosil C18 resin is a silica-based stationary phase material used in liquid chromatography. It is composed of silica particles with chemically bonded C18 alkyl chains, providing a hydrophobic surface for the separation of a wide range of organic compounds. The core function of Reprosil C18 resin is to serve as a reliable and efficient stationary phase in reversed-phase liquid chromatography systems.

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Lab products found in correlation

Easy nlc 1200 system, by Thermo Fisher Scientific (3 mentions) Ultimate 3000, by Thermo Fisher Scientific (2 mentions) Orbitrap lumos tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions) Orbitrap elite, by Thermo Fisher Scientific (1 mentions) Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions) Nanospray flex ion source, by Thermo Fisher Scientific (1 mentions) Q exactive plus, by Thermo Fisher Scientific (1 mentions) Orbitrap xl, by Agilent Technologies (1 mentions) Zorbax 300sb c18, by Agilent Technologies (1 mentions)

8 protocols using reprosil c18 resin

Proteomic Analysis by LC-MS/MS on Orbitrap

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LC-MS/MS was performed on a Lumos Tribrid Orbitrap Mass Spectrometer (ThermoFisher, San Jose, CA) equipped with Ultimate 3000 (Thermo-Fisher, CA) nano-HPLC. Peptides were separated onto a 150 μm inner diameter microcapillary trapping column packed with approximately 2cm of C18 Reprosil resin (5 μm, 100 Å, Dr. Maisch GmbH, Germany) followed by a PharmaFluidics (Gent, Belgium) 50 cm analytical column. Separation was achieved through applying a gradient from 5– 27% acetonitrile in 0.1% formic acid over 90 min at 200 nL min⁻¹. Electrospray ionization was enabled by applying a voltage of 2 kV using a home-made electrode junction at the end of the microcapillary column and sprayed from metal tips (PepSep, Denmark). The mass spectrometry survey scan was performed in the Orbitrap in the range of 400–1,800 m/z at a resolution of 6 × 10⁴, followed by the selection of the twenty most intense ions (TOP20) for CID-MS2 fragmentation in the Ion trap using a precursor isolation width window of 2 m/z, AGC setting of 10,000, and a maximum ion accumulation of 100 ms. Singly charged ion species were not subjected to CID fragmentation. Normalized collision energy was set to 35 V and an activation time of 10 ms. Ions in a 10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60 seconds.

Wong P.C., Waggoner D., Subramaniam J.R., Tessarollo L., Bartnikas T.B., Culotta V.C., Price D.L., Rothstein J, & Gitlin J.D. (2000). Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase. Proceedings of the National Academy of Sciences of the United States of America, 97(6), 2886-2891.

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LC-MS/MS Analysis of Peptides

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The LC-MS/MS experiment was performed on a Lumos Tribrid Orbitrap Mass Spectrometer (Thermo Fischer) equipped with Ultimate 3000 (Thermo Fisher) nano-HPLC. Peptides were separated onto a 150μm inner diameter microcapillary trapping column packed first with approximately 2cm of C18 Reprosil resin (5μm, 100 Å, Dr. Maisch GmbH, Germany) followed by PharmaFluidics (Gent, Belgium) 50cm analytical column. Separation was achieved by applying a gradient from 5–27% acetonitrile in 0.1% formic acid over 90 min at 200 nl/min. Electrospray ionization was enabled by applying a voltage of 2 kV using a homemade electrode junction at the end of the microcapillary column and sprayed from metal tips (PepSep, Denmark). The mass spectrometry survey scan was performed in the Orbitrap in the range of 400–1,800 m/z at a resolution of 6×10⁴, followed by the selection of the twenty most intense ions (TOP20) for CID-MS2 fragmentation in the Ion trap using a precursor isolation width window of 2 m/z, AGC setting of 10,000, and a maximum ion accumulation of 100 ms. Singly charged ion species were not subjected to CID fragmentation. The normalized collision energy was set to 35 V and an activation time of 10 ms. Ions in a 10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60s.

Moghimi S., Viktorova E.G., Gabaglio S., Zimina A., Budnik B., Wynn B.G., Sztul E, & Belov G.A. (2022). A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication. PLOS Pathogens, 18(10), e1010906.

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Nanoflow LC-MS/MS Proteomic Analysis

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All
samples were unfractionated unless otherwise indicated. Samples were
loaded to self-pulled (P2000 Sutter Laser puller, Sutter Instrument,
Novato, CA) 360 μm OD × 75 μm ID 10 cm columns with
a 10 μm tip and packed with 3 μm Reprosil C18 resin (Dr.
Maisch, Germany) using a pressure cell (NextAdvance, Averill Park,
NY). Peptides were analyzed with 90 min gradients of 3–35%
acetonitrile at 200 nL/min nanoLC–MS (Thermo Dionex RSLCnano,
Sunnyvale, CA) on an Orbitrap Elite (Thermo, Bremen Germany). Orbitrap
FTMS spectra (R = 30 000 at 400 m/z; m/z 350–1600;
3e6 target; max 500 ms ion injection time) and Top15 data dependent
CID MS/MS spectra (1e4 target; max 250 ms injection time) were collected
with dynamic exclusion for 180 s and an exclusion list size of 500.
The normalized collision energy applied for CID was 35% for 10 ms.

Lau H.T., Suh H.W., Golkowski M, & Ong S.E. (2014). Comparing SILAC- and Stable Isotope Dimethyl-Labeling Approaches for Quantitative Proteomics. Journal of Proteome Research, 13(9), 4164-4174.

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Quantitative Proteomics by LC-MS/MS

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LC/MS was performed on a Thermo Scientific™ Q Exactive Plus equipped with an ultra-high performance liquid chromatography (UHPLC) unit (Thermo Scientific Dionex Ultimate 3000) and a Nanospray Flex Ion-Source (Thermo Fisher Scientific). Peptides were separated on a column with a 2.4 μm Reprosil C18 resin (Dr. Maisch GmbH) in-house packed picotip emitter tip (diameter 100 μm, 15 cm from New Objectives) with a gradient from 4% acetonitrile, 0.1% formic acid to 60% eluent B (99% acetonitrile, 0.1% formic acid) in two steps for 35 min. MS data were recorded by data-dependent acquisition. Peptides and proteins were identified and quantified using PEAKS 7 Studio (Bioinformatics Solutions Inc. Waterloo, Canada), human reference proteome set (UniProt, May 2020, 74 823 entries, supplemented with SRSF6–GFP) with a false discovery rate (FDR) <1%. Oxidation of methionine (+15.99), deamidation on asparagine and glutamine (+0.98), phosphorylation on serine, threonine and tyrosine (+79.97) and carbamidomethylation (+57.02) on cysteine were selected as variable.

de Oliveira Freitas Machado C., Schafranek M., Brüggemann M., Hernández Cañás M.C., Keller M., Di Liddo A., Brezski A., Blümel N., Arnold B., Bremm A., Wittig I., Jaé N., McNicoll F., Dimmeler S., Zarnack K, & Müller-McNicoll M. (2023). Poison cassette exon splicing of SRSF6 regulates nuclear speckle dispersal and the response to hypoxia. Nucleic Acids Research, 51(2), 870-890.

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Peptide Fractionation and Mass Spectrometry

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Peptides were eluted from StageTips using elution buffer (40% acetonitrile, 1% FA) and then loaded on a self-pulled 360 μm OD × 100 μm ID 20 cm column with a 7 μm tip packed with 3 μm Reprosil C18 resin (Dr. Maisch, Germany). For pull-down experiment, peptides were analyzed by nanoLC-MS in a 90 min gradient from 15% to 38% buffer B (for phosphopeptides 6%–35% buffer B) at 300 nL/min using a Thermo EASY nLC 1200 system (buffer A: 0.1% acetic acid; buffer B: 0.1% acetic acid, 80% acetonitrile). Mass spectra were collected from an Orbitrap Fusion Lumos Tribrid Mass Spectrometer using the following settings. For MS1, Orbitrap FTMS (R = 60 ,000 at 200 m/z; m/z 350–1600; 7e5 target; max 20 ms ion injection time); For MS2, Top Speed data-dependent acquisition with 3 s cycle time was used, HCD MS2 spectra were collected using the Orbitrap mass analyzer(R = 30,000 at 200 m/z; 31% CE; 5e4 target; max 100 ms injection time) an intensity filter was set at 2.5e4 and dynamic exclusion for 45 s.

Omar M.H., Byrne D.P., Jones K.N., Lakey T.M., Collins K.B., Lee K.S., Daly L.A., Forbush K.A., Lau H.T., Golkowski M., McKnight G.S., Breault D.T., Lefrançois-Martinez A.M., Martinez A., Eyers C.E., Baird G.S., Ong S.E., Smith F.D., Eyers P.A, & Scott J.D. (2022). Mislocalization of protein kinase A drives pathology in Cushing’s syndrome. Cell reports, 40(2), 111073.

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Peptide Fractionation and Mass Spectrometry

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Peptide Purification and Mass Spectrometry

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Peptides were eluted from StageTips using elution buffer (40% acetonitrile, 1% FA) and then loaded on a self-pulled 360 mm OD × 100 mm ID 20 cm column with a 7 μm tip packed with 3 μm Reprosil C18 resin (Dr. Maisch, Germany). For pull-down experiment, peptides were analyzed by nanoLC-MS in a 90 min gradient from 15% to 38% solvent B (for phosphopeptides 6%–35% solvent B) at 300 nL/min using a Thermo EASY nLC 1200 system (solvent A: 0.1% acetic acid; solvent B: 0.1% acetic acid, 80% acetonitrile). Mass spectra were collected from an Orbitrap Fusion Lumos Tribrid Mass Spectrometer using the following settings. For MS1, Orbitrap FTMS (R = 60,000 at 200 m/z; m/z 350–1600; 7e5 target; max 20 ms ion injection time); For MS2, Top Speed data-dependent acquisition with 3 s cycle time was used, HCD MS2 spectra were collected using the Orbitrap mass analyzer(R = 30,000 at 200 m/z; 31% CE; 5e4 target; max 100 ms injection time) an intensity filter was set at 2.5e4 and dynamic exclusion for 45 s.

Lauer S.M., Omar M.H., Golkowski M.G., Kenerson H.L., Lee K.S., Pascual B.C., Lim H.C., Forbush K., Smith F.D., Gordan J.D., Ong S.E., Yeung R.S, & Scott J.D. (2024). Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma. Cell reports, 43(2), 113678.

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Quantitative Mass Spectrometry Analysis

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LC/MS was performed on a Thermo Scientific Orbitrap XL mass spectrometer linked to an Agilent1200 nano-high-performance liquid chromatography. Peptides were resolved in 4% acetonitrile and 0.5% formic acid and loaded on a C18 reversed-phase precolumn (Zorbax 300SB-C18; Agilent Technologies, Palo Alto, CA) followed by separation on an in-house packed 3 mm Reprosil C18 resin (Dr. Maisch GmbH, Ammerbuch, Germany) picotip emitter tip (diameter 75 mm, length 10 cm; New Objectives, Woburn, MA) using a gradient from 5% acetonitrile, 0.1% formic acid to 50% acetonitrile, 0.1% formic acid for 90 minutes with a flow rate of 200 nL/min. MS data were recorded by data-dependent Top10 acquisition selecting the 10 most abundant precursor ions in positive mode for fragmentation using dynamic exclusion of 3 minutes. Only highly charged ions (21) were selected for MS/MS scans in the linear ion trap by CID at 35% collision energy. Lock mass option for m/z 445.120,025 was enabled to ensure high mass accuracy during many following runs. 57

Kallenborn-Gerhardt W., Möser C.V., Lorenz J.E., Steger M., Heidler J., Scheving R., Petersen J., Kennel L., Flauaus C., Lu R., Edinger A.L., Tegeder I., Geisslinger G., Heide H., Wittig I, & Schmidtko A. (2017). Rab7-a novel redox target that modulates inflammatory pain processing. Pain, 158(7).

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