Pepmap rslc column

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PepMap RSLC column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of peptides. The column features a C18 stationary phase and is suitable for nano-flow and micro-flow chromatography applications.

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11 protocols using pepmap rslc column

Cross-linking and Mass Spectrometry Analysis

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Chemical cross-linking reactions were carried out following the manufacturer’s instruction. PDE6 complexes were buffer-exchanged into HEPES buffer (20 mM HEPES, 100 mM NaCl, 5mM MgCl₂, pH 8.0) and cross-linked with 200-fold (or 70-fold) molar excess of DSS cross-linker at room temperature for 1 h. After the cross-linking reaction was quenched with 1 μL 0.8M ammonium hydroxide, proteins were concentrated by rotary evaporation under vacuum, separated by SDS-PAGE, and visualized with Coomassie Brilliant Blue G-250.
Cross-linked products were in-gel digested and analyzed by LC-MS and LC-MS-MS as described previously [42 (link)]. Briefly, 1μl aliquot of the digestion mixture was injected into a Dionex Ultimate 3000 RSLCnano UHPLC system (Dionex Corporation, Sunnyvale, CA), and separated by a 75 μm × 25 cm PepMap RSLC column (100 Å, 2 μm) at a flow rate of ~450 nl/min. The eluant was connected directly to a nanoelectrospray ionization source of an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA). LC-MS data were acquired in an information-dependent acquisition mode, cycling between a MS scan (m/z 315–2,000) acquired in the Orbitrap, followed by low-energy CID analysis on the 3 most intense multiply charged precursors acquired in the linear ion trap.

Chu F., Hogan D., Gupta R., Gao X.Z., Nguyen H.T, & Cote R.H. (2019). Allosteric regulation of rod photoreceptor phosphodiesterase 6 (PDE6) elucidated by chemical cross-linking and quantitative mass spectrometry. Journal of molecular biology, 431(19), 3677-3689.

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Proteomic Analysis of Chromatophore Cells

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The yellow, red or brown chromatophore cells were lysed by sample buffer and sonicated to solubilize proteins. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue G250.
Each lane of different color chromatophore cells were divided into five bands, reduced and alkylated, digested in-gel with trypsin. Tryptic peptides were extracted with 50% acetonitrile in 2% formic acid. Volume of the digestion mixture was reduced to 3.5 µL, and 1 µL was injected in a Dinonex Ultimate 3000 RSLC nano UHPLC system (Dionex Corporation, Sunnyvale, CA), and separated by a 75 µm x 25 cm PepMap RSLC column (100 Å, 2 µm) at a flow rate of ~450 nL min⁻¹. The eluant was connected directly to a nanoelectrospray ionization source of an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA). LC-MS data were acquired in an information-dependent acquisition mode, cycling between a MS scan (m/z = 315–2000) acquired in the Orbitrap, followed by low-energy CID analysis on three most intense multiply charged precursors acquired in the linear ion trap.
For the granules, pigment, and extracted granules, the samples were denatured and separated by SDS-PAGE. After staining the gel with Coomassie blue G250, visible bands from all three samples were excised for in-gel digestion and MS/MS analysis as above.

Williams T.L., Senft S.L., Yeo J., Martín-Martínez F.J., Kuzirian A.M., Martin C.A., DiBona C.W., Chen C.T., Dinneen S.R., Nguyen H.T., Gomes C.M., Rosenthal J.J., MacManes M.D., Chu F., Buehler M.J., Hanlon R.T, & Deravi L.F. (2019). Dynamic pigmentary and structural coloration within cephalopod chromatophore organs. Nature Communications, 10, 1004.

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PDE6 Holoenzyme Cross-linking Analysis

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Chemical cross-linking reactions were carried out, following the Pierce instruction for each cross-linker. PDE6 holoenzyme was buffer-exchanged into either HEPES buffer (20 mM HEPES, 100 mM NaCl, 5mM MgCl₂, pH 8.0) for BS3, DSS, BS(PEG)5, BS(PEG)9 and Sulfo-MBS cross-linking; or MES buffer (100 mM MES, pH 6.5) for EDC cross-linking. The PDE6 holoenzyme was cross-linked with ~50-fold molar excess of the cross-linker. After the cross-linking reaction was quenched, proteins were precipitated with trichloroacetic acid, separated by SDS-PAGE and visualized with Coomassie brilliant blue G-250.
Cross-linked products were in-gel digested and analyzed by LC-MS and LC-MS-MS as described previously (53 (link)). Briefly, l μ1 aliquot of the digestion mixture was injected into an Dionex Ultimate 3000 RSLCnano UHPLC system (Dionex Corporation, Sunnyvale, CA), and separated by a 75 μm × 25 cm PepMap RSLC column (100 Å, 2 μm) at a flow rate of ~450 nl/min. The eluant was connected directly to a nanoelectrospray ionization source of an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA). LC-MS data were acquired in an information-dependent acquisition mode, cycling between a MS scan (m/z 315-2,000) acquired in the Orbitrap, followed by low-energy CID analysis on 3 most intense multiply charged precursors acquired in the linear ion trap.

Zeng-Elmore X., Gao X.Z., Pellarin R., Schneidman-Duhovny D., Zhang X.J., Kozacka K.A., Tang Y., Sali A., Chalkley R.J., Cote R.H, & Chu F. (2014). Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling. Journal of molecular biology, 426(22), 3713-3728.

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HLA Ligand Profiling by Nanoscale LC-MS

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HLA ligand extracts were analyzed in five technical replicates as described previously [31 (link)]. In brief, peptide samples were separated by nanoflow HPLC (RSLCnano, Thermo Fisher, Waltham, MA, USA) using a 50 μm×25 cm PepMap RSLC column (Thermo Fisher) and a gradient ranging from 2.4 to 32.0% acetonitrile over the course of 90 min. Eluting peptides were analyzed in an online-coupled LTQ Orbitrap XL mass spectrometer (Thermo Fisher) using a top 5 CID (collision-induced dissociation) fragmentation method.

Backert L., Kowalewski D.J., Walz S., Schuster H., Berlin C., Neidert M.C., Schemionek M., Brümmendorf T.H., Vucinic V., Niederwieser D., Kanz L., Salih H.R., Kohlbacher O., Weisel K., Rammensee H.G., Stevanović S, & Walz J.S. (2017). A meta-analysis of HLA peptidome composition in different hematological entities: entity-specific dividing lines and “pan-leukemia” antigens. Oncotarget, 8(27), 43915-43924.

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HLA Ligand Analysis by Nano-HPLC-MS

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HLA ligand extracts were analyzed in five technical replicates as described previously.^{23 (link)} In brief, peptide samples were separated by nanoflow HPLC (RSLCnano, Thermo Fisher, Waltham, MA, USA) using a 50 μm × 25 cm PepMap RSLC column (Thermo Fisher) and a gradient ranging from 2.4 to 32.0% acetonitrile over the course of 90 min. Eluting peptides were analyzed in an online coupled LTQ Orbitrap XL mass spectrometer (Thermo Fisher) using a top five CID (collision-induced dissociation) fragmentation method.

Kowalewski D.J., Walz S., Backert L., Schuster H., Kohlbacher O., Weisel K., Rittig S.M., Kanz L., Salih H.R., Rammensee H.G., Stevanović S, & Stickel J.S. (2016). Carfilzomib alters the HLA-presented peptidome of myeloma cells and impairs presentation of peptides with aromatic C-termini. Blood Cancer Journal, 6(4), e411-.

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Peptide Separation and Analysis by Nano-UPLC-MS

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Peptides were injected into an Ultimate 3000 RSLC system (Thermo Scientific, Sunnyvale, California, USA) connected to a Q Exactive HF equipped with a nanospray Flex ion source (Thermo Scientific, Bremen, Germany). The samples were loaded on an Acclaim PepMap 100, 2cm x 75μm i.d. nanoViper pre-column, packed with 3μm C18 beads at a flow rate of 5μl/min for 5 min with 0.1% TFA (trifluoroacetic acid, vol/vol). Peptides were separated during a biphasic ACN gradient from two nanoflow UPLC pumps (flow rate of 0.250 μl/min) on a 50 cm analytical column (Easy-Spray 803, 50cm x 75μm i.d. PepMap RSLC column, packed with 2μm C18 beads (Thermo Scientific). Solvent A was 0.1% FA (vol/vol) in water, and solvent B was 100% ACN, 0.1% FA. The following gradient was used; 0 min 5% B, 5 min 5% B, 140 min 35% B, 155 min 80% B, 170 min 80% B, 175 min 5% B, 195 min 5% B. Samples were a maximum of 48 hours in the autosampler at 10˚C prior to injection.

Lereim R.R., Nytrova P., Guldbrandsen A., Havrdova E.K., Myhr K.M., Barsnes H, & Berven F.S. (2024). Natalizumab promotes anti-inflammatory and repair effects in multiple sclerosis. PLOS ONE, 19(3), e0300914.

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Tryptic Peptide Separation and Mass Spectrometry

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The tryptic peptides were dissolved
in 10 μL of 4% acetonitrile, 0.1% formic acid, and 1.5 μL
was loaded and separated on an Acclaim PepMap RSLC column (75 μm
× 25 cm, C18, 2 μm, 100 Å, Thermo) with a 50 min 5–35%
linear gradient of mobile phase B (80% aqueous acetonitrile containing
0.1% formic acid) in mobile phase A (0.1% formic acid in water), followed
by a 10 min isocratic 95% B. The gradient was delivered by an Easy-nLC
1200 system (Thermo) at 300 nL/min. An Orbitrap Eclipse mass spectrometer
(Thermo Scientific) data acquisition method was based on the “Single
Cell LFQ” template with the following modifications: cycle
time 2 s, maximum injection time for MS2 250 ms, charge states 2–6.

Chauhan S.S., Marotta N.J., Karls A.C, & Weinert E.E. (2022). Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation. ACS Central Science, 8(11), 1518-1526.

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Peptide Digestion and Mass Spectrometry Analysis

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Dried peptide digests were resuspended in a solution of 5% ACN, 0.1% formic acid, and 0.005% trifluoroacetic acid (20 uL). Samples were separated by ultra-high-pressure reverse phase chromatography using an Acclaim PepMap RSLC column and an Easy nLC 1000 UHPLC system (Thermo). Peptides were analyzed with a Fusion mass spectrometer (Thermo) with a 120,000 resolution orbitrap MS1 scan over the range of m/z 375–1600, followed by ion trap resolution MS2 scans using an m/z 1.6 window and 30% normalized collision energy for HCD. Peak lists were generated with Proteome Discoverer (version 1.4; Thermo), and peptides scored using Mascot (version 2.4; Matrix Science) and Sequest (version 2.1). Label-free quantitation was performed using MaxQuant software.

Case K.C., Beltman R.J., Pflum M.K, & Greenberg M.L. (2023). Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase. Scientific Reports, 13, 14844.

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Proteomics Analysis of Cross-linked Peptides

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The dried samples were resuspended in 20 μl of water/acetonitrile/formic acid (95:5:0.1, v/v/v) and 5 μl were analysed by LC−MS/MS on an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific) equipped with a Nanoflex electrospray source coupled to an Easy nLC 1200 HPLC system (ThermoFisher Scientific). The cross-linked peptides were separated on a PepMap RSLC column (250 mm × 75 μm, 2 μm particle size, ThermoFisher Scientific). Chromatographic separation was performed with a 60 min gradient starting at 6% and increasing to 40% B (mobile phase A: water/acetonitrile/formic acid (98:2:0.15, v/v/v); mobile phase B: acetonitrile/water/formic acid (80:20:0.15, v/v/v)), with a flow rate at 300 nl/min. The Orbitrap Fusion Lumos was operated in positive ion, data-dependent acquisition mode. Acquisition was performed at a resolution of 120 000 in 3 s cycles. During each cycle, precursor ions were selected for fragmentation using stepped higher energy collision-induced dissociation (normalized collision energy, 23 ± 5%). Fragment ions were detected in the Orbitrap at a resolution of 30 000, with an isolation window of 1.2 m/z, a dynamic exclusion duration of 30 s and selected charge states = 2–7⁺.

Padroni G., Bikaki M., Novakovic M., Wolter A.C., Rüdisser S.H., Gossert A.D., Leitner A, & Allain F.H. (2023). A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2. Nucleic Acids Research, 51(9), 4555-4571.

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Tandem Mass Spectrometry Peptide Analysis

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Tandem mass spectrometry analysis was performed as described previously.²⁷ (link) In brief, reconstituted peptides (20 µl mobile phase A: 2% v/v acetonitrile, 0.1% v/v formic acid) were analyzed using a EASY-nLC 1000 nano-HPLC system connected to a LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). For peptide separation, a PepMap RSLC column (75 μm ID, 150 mm length, C18 stationary phase with 2 µm particle size and 100 Å pore size, Thermo Fisher Scientific) was used, running a gradient from 5% to 35% mobile phase B (98% v/v acetonitrile, 0.1% v/v formic acid) at a flow rate of 300 nl/min. For data-dependent acquisition, up to 10 precursors from a MS1 scan (resolution = 60,000) in the range of m/z 250-1800 were selected for collision-induced dissociation (CID: 10 ms, 35% normalized collision energy, activation q of 0.25).

Schmidtmann E., Anton T., Rombaut P., Herzog F, & Leonhardt H. (2016). Determination of local chromatin composition by CasID. Nucleus, 7(5), 476-484.

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