Pepmap rslc easy spray column

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The PepMap RSLC EASY-Spray column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of peptides. It features a reversed-phase stationary phase and is compatible with the EASY-Spray ionization source for mass spectrometry applications.

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EASY-Spray PepMap RSLC C18 column EASY-Spray PepMap RSLC C18 75 µm × 250 mm column EASY-Spray PepMap RSLC C18 75μm × 250mm column Easy-Spray PepMap® RSLC Easy Spray PepMap column PepMap RSLC column EASY-Spray column PepMap RSLC EASY-Spray Easy Column

10 protocols using pepmap rslc easy spray column

Synaptosome Proteome Extraction and Analysis

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Lipids were removed from synaptosome samples (n = 5 + 5 mice/genotype) as previously described (Gorski et al., 2020 (link)). Briefly, samples were incubated overnight at −20°C in five volumes ice-cold (−20°C) acetone and centrifuged twice at 1000 × g for 10 min at +4°C with an additional acetone wash in between. Pellets were air dried for 5 min and resuspended in freshly prepared 6.0 M urea/25 mM ammonium bicarbonate. Protein concentrations (μg/μl) were determined spectrophotometrically using the BCA protein assay kit (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions.
For proteome analysis, 5 μg of each sample was reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAA), and the urea concentration was diluted to 1 M, followed by overnight digestion with trypsin (Promega Corporation, WI, USA) at +37°C. Peptides were desalted and concentrated by the STAGE-TIP method using a C18 resin disk (3 M Empore). Samples were eluted with 0.1% formic acid/60% acetonitrile, dried, and solubilized in 7 μl 0.1% formic acid prior to mass spectrometry analysis. Each peptide mixture was analyzed using an EASY-nLC system coupled to the QExactive Plus mass spectrometer (ThermoElectron, Bremen, Germany) equipped with the EASY Spray PepMap®RSLC column (C18, 2 μl, 100 Å, 75 μm x 25 cm) using a 120 min LC separation gradient.

Gorski K., Jackson C.B., Nyman T.A., Rezov V., Battersby B.J, & Lehesjoki A.E. (2023). Progressive mitochondrial dysfunction in cerebellar synaptosomes of cystatin B-deficient mice. Frontiers in Molecular Neuroscience, 16, 1175851.

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Co-IP Protein Digestion and Mass Spectrometry

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Beads from the Co-IP were dissolved in 20 µL 0.2 % ProteaseMAX Surfactant, Trypsin Enhancer in 50 mM NH4HCO3 followed by protein reduction, alkylation and on-beads digestion with trypsin overnight in 37 °C. After digestion, the samples were centrifuged at 14 000 x g for 10 min, trypsin was inactivated by adding 100 µl 1 % TFA, and the samples were again centrifuged at 14 000 x g for 10 min. The resulting peptides were desalted and concentrated before mass spectrometry by the STAGE-TIP method using a C18 resin disk (3M Empore). Each peptide mixture was analyzed by a nEASY-LC coupled to QExactive Plus (ThermoElectron, Bremen, Germany) with EASY Spray PepMap®RSLC column (C18, 2 µl, 100Å, 75 µm x 50 cm). The resulting MS raw files were submitted for protein identification using Proteome Discoverer 2.1 (Thermo Fisher Scientific) and Mascot 2.

Olsen P.A., & Krauß S. (2021). The adenoviral E1B-55k protein present in HEK293 cells mediates abnormal accumulation of key WNT signaling proteins in large cytoplasmic aggregates.

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LC-MS/MS Peptide Analysis Protocol

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LC-MS/MS of peptide fractions was performed on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) coupled to a Q Exactive Orbitrap mass spectrometer equipped with EASY-Spray ion source (Thermo Fisher Scientific). Peptide samples were trapped on an Acclaim PepMap trap column (C18, 3 µm, 100 Å, 75 µm × 20 mm) and separated on an EASY-Spray PepMap RSLC column (C18, 2 µm, 100 Å, 75 µm × 50 cm, Thermo Fisher Scientific) using a gradient of A (0.1% formic acid) and B (95% MeCN, 0.1% formic acid), ranging from 3% to 40% B in 50 min with a flow of 0.25 µL/min. The top 10 ions were selected for fragmentation, and the survey scan was performed at 70,000 resolution from 300 to 1,600 m/z, with a max injection time of 100 ms and a target of 1 × 10⁶ ions. For generation of high energy collision dissociation (HCD) fragmentation spectra, a max ion injection time of 200 ms and AGC of 2 × 10⁵ were used before fragmentation at 30% normalized collision energy, 17,500 resolution. Precursors were isolated with a width of 2 m/z and put on the exclusion list for 30 s. Single and unassigned charge states were rejected from precursor selection.

Bergqvist F., Ossipova E., Idborg H., Raouf J., Checa A., Englund K., Englund P., Khoonsari P.E., Kultima K., Wheelock C.E., Larsson K., Korotkova M, & Jakobsson P.J. (2019). Inhibition of mPGES-1 or COX-2 Results in Different Proteomic and Lipidomic Profiles in A549 Lung Cancer Cells. Frontiers in Pharmacology, 10, 636.

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LC-MS/MS Analysis of Peptides

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Before LC-MS/MS analysis, the samples were desalted by reversed-phase chromatography using C18 microcolumns prepared by stacking three layers of C18 Empore Extraction Disk (Varian, St. Paul, MN) into 200-mL pipette tips. For samples from patients P1 to P10 ( 2) were analyzed by similar conditions as described above; the instrument was an Q Exactive hybrid quadropole-orbitrap (Thermo Fisher Scientific), and peptide separation was performed on a 25-cm EASY Spray PepMapRSLC column (C18, 2 mm, 100 Å, 75-mm inner diameter; Thermo Fisher Scientific) using a 300-minute gradient, with 2% to 30% B in 300 minutes. Isolation window was set to 3.0 m/z for MS/MS scans.

Tutturen A.E.V., Dørum S., Clancy T., Reims H.M., Christophersen A., Lundin K.E.A., Sollid L.M., de Souza G.A, & Stamnaes J. (2018). Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry. The American journal of pathology, 188(7).

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Mass Spectrometry Profiling of Mussel Extracts

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Profiling of the mussel extracts was performed as previously described for secretome samples (41 (link)). Briefly, 100 µg of crude and clarified mussel extracts were enzymatically digested using a trypsin/Lys-C mixture, followed by desalting and purification using STop And Go Extraction (STAGE)-tips (42 (link)). Samples were measured on an Orbitrap Exploris 240 hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 high-performance liquid chromatography device (Thermo Fisher Scientific). Samples were loaded onto an in-line 75 mm by 50 cm PepMap RSLC EASY-Spray column filled with 2-mm C18 reverse-phase silica beads (Thermo Fisher Scientific). Separated peptides were electro-sprayed into the mass spectrometer with a linear gradient of 3–20% buffer B (80% acetonitrile and 0.5% acetic acid) over a 60-min gradient, followed by a wash with 100% buffer B with a 250 nL/min flow rate. The mass spectrometer was operated in a data-dependent acquisition model and switched between one full scan and MS/MS scans of abundant peaks. Full scans (m/z 400 to 2,000) were acquired in the Orbitrap mass analyzer with a resolution of 120,000 at m/z 200.

Gutierrez-Gongora D., Woods M., Prosser R.S, & Geddes-McAlister J. (2024). Natural compounds from freshwater mussels disrupt fungal virulence determinants and influence fluconazole susceptibility in the presence of macrophages in Cryptococcus neoformans. Microbiology Spectrum, 12(3), e02841-23.

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Sensitive Peptide Separation and Analysis

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Following trypsin digestion, peptides were separated by on-line reverse-phase high-pressure liquid chromatography and mass spectrometry (LC-MS) using a LTQ-Orbitrap Elite (linear ion trap-orbitrap) hybrid analyzer outfitted with a nanospray source and EASY-SPRAY 1200 split free nano-LC system (ThermoFischer Scientific). A 50 cm PepMap RSLC Easy-Spray column filled with 2 µm C18 beads was used in the HPLC (ThermoFischer Scientific). The resultant peptides were loaded at a pressure of 800 BAR and eluted over 0–120 min at a rate of 250 nL/min using a 0–35% acetonitrile gradient in 0.1% formic acid. In the mass spectrometer, one full MS Scan (400–1500 m/z) was performed in the Orbitrap Elite with an automatic gain control (AGC) of 500,000, max ion time of 200 ms, and one microscan, at a resolution of 240,000. Ten data-dependent MS/MS scans were performed in the linear ion trap using the ten most intense ions at 35% normalized collision energy. MS and MS/MS scans were done in parallel. For MS/MS scans, the AGC was 10,000 with a maximum ion injection time of 100 ms. A minimum ion intensity of 1000 was required to trigger the MS/MS scan. Dynamic exclusion was applied with a maximum exclusion list of 500, with one repeat count and a repeat duration of 8 s and exclusion duration of 30 s.

Abdelmegid S., Murugaiyan J., Abo-Ismail M., Caswell J.L., Kelton D, & Kirby G.M. (2017). Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis. International Journal of Molecular Sciences, 19(1), 78.

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LC-MS/MS Peptide Separation and Analysis

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For liquid chromatography-tandem mass spectrometry (LC-MS/MS), digested peptides were resuspended in buffer A (0.1% formic acid) and analyzed on a Orbitrap Exploris 240 hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 high-performance liquid chromatography device (Thermo Fisher Scientific). Samples were loaded onto an in-line 75-μm by 50-cm PepMap RSLC EASY-Spray column filled with 2-μm C₁₈ reverse-phase silica beads (Thermo Fisher Scientific). Separated peptides were electrosprayed into the mass spectrometer with a linear gradient of 3% to 20% buffer B (80% acetonitrile, 0.5% acetic acid) over a 3-h gradient, followed by a wash with 100% buffer B with a 250-nL/min flow rate. The mass spectrometer switched between one full scan and MS/MS scans of abundant peaks. Full scans (m/z 400 to 2,000) were acquired in the Orbitrap mass analyzer with a resolution of 120,000 at 200 m/z 200.

Sukumaran A., Ball B., Krieger J.R, & Geddes-McAlister J. (2022). Cross-Kingdom Infection of Macrophages Reveals Pathogen- and Immune-Specific Global Reprogramming and Adaptation. mBio, 13(4), e01687-22.

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High-resolution Proteomics Workflow with Orbitrap

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Samples were run over a 60-min chromatographic gradient with an Orbitrap Exploris 240 hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 high-performance liquid chromatography device (Thermo Fisher Scientific). Samples were loaded onto an in-line 75-mm by 50-cm PepMap RSLC EASY-Spray column filled with 2-mm C18 reverse-phase silica beads (Thermo Fisher Scientific). Separated peptides were electro-sprayed into the mass spectrometer with a linear gradient of 3% to 20% buffer B (80% acetonitrile, 0.5% acetic acid) over a 3 h gradient, followed by a wash with 100% buffer B with a 250-nL/min flow rate. The mass spectrometer switched between one full scan and MS/MS scans of abundant peaks. Full scans (m/z 400 to 2,000) were acquired in the Orbitrap mass analyzer with a resolution of 120,000 at m/z 200.

Gutierrez-Gongora D., Raouf-Alkadhimi F., Prosser R.S, & Geddes-McAlister J. (2023). Differentiated extracts from freshwater and terrestrial mollusks inhibit virulence factor production in Cryptococcus neoformans. Scientific Reports, 13, 4928.

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Peptide Identification by Nano-LC-MS/MS

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For the identification of the peptides a Dionex Ultimate 3000 RSLCnano System coupled to an Orbitrap Eclipse Tribrid Mass Spectrometer (both Thermo Fisher Scientific, Vienna, Austria) was used. The dried samples were dissolved in 25 µL mobile phase A (98% H₂O, 2% ACN, 0.1% FA), shaken for 10 min at 30 °C and centrifuged at 14000 g for 1 min. 2 µL were injected onto a PepMap RSLC EASY-Spray column (C18, 2 µm, 100 Å, 75 µm x 15 cm, Thermo Fisher Scientific). Separation occurred at 300 nL min⁻¹ with a flow gradient from 2-35% mobile Phase B (2% H₂O, 98% ACN, 0.1% FA) within 25 min resulting in a total method time of 55 min. Mass spectrometer was operated with the FAIMS Pro System in positive ionization mode at alternating CV −60 and −75. The scan range was 350-2000 m z⁻¹ using a resolution of 60,000 @200 m z⁻¹ on MS1 level. Isolated peptides were fragmented using CID at a collision energy of 30% and fragments were analyzed in the Orbitrap with a resolution of 30,000 @200 m z⁻¹.

Dereli I., Telychko V., Papanikos F., Raveendran K., Xu J., Boekhout M., Stanzione M., Neuditschko B., Imjeti N.S., Selezneva E., Tuncay H., Demir S., Giannattasio T., Gentzel M., Bondarieva A., Stevense M., Barchi M., Schnittger A., Weir J.R., Herzog F., Keeney S, & Tóth A. (2024). Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes. Nature Communications, 15, 2941.

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Peptide Identification by Mass Spectrometry

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Digested peptides were resuspended in 12 μl buffer A (0.1% TFA). Six μl of each sample was analyzed on a Q Exactive™ HF-X hybrid quadrupole-orbitrap mass spectrometer (ThermoFisher Scientific) coupled to an Easy-nLC™ 1200 High-Performance Liquid Chromatography (ThermoFisher Scientific). Samples were loaded onto an in-line 75 μm × 50 cm PepMap RSLC EASY-Spray column filled with 2 μm C18 reverse-phase silica beads (ThermoFisher Scientific). Peptides were separated and directly electrosprayed into the mass spectrometer using a linear gradient from 3 to 20% Buffer B over 18 min, from 20 to 35% Buffer B over 31 mins, followed by a steep 2 min ramp to 100% Buffer B for 9 min in 0.1% Formic acid at a constant flow of 250 nl/min. The mass spectrometer was operated in data-dependent mode, switching automatically between one fill scan and subsequent MS/MS scans of 30 most abundant peaks, with full-scans (m/z 400–1600) acquired in the Orbitrap analyzer with a resolution of 60,000 at 400 m/z.

Sukumaran A., Pladwig S, & Geddes-McAlister J. (2021). Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production. BMC Microbiology, 21, 43.

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