Acclaim pepmap rslc analytical column

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Acclaim PepMap RSLC analytical 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 suitable for use in RSLC (Rapid Separation Liquid Chromatography) systems.

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Acclaim PepMap RSLC (134 citations) Acclaim PepMap RSLC column (45 citations) Acclaim PepMap (38 citations) PepMap RSLC (19 citations) PepMap RSLC column (11 citations) Acclaim PepMap RSLC C18 analytical column (9 citations) Acclaim PepMap analytical column (5 citations) Analytical columns (4 citations)

16 protocols using acclaim pepmap rslc analytical column

Nano-LC-MS/MS Proteomic Analysis Protocol

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Using a Dionex UltiMate 3000 RSLCnano system equipped with a Dionex UltiMate 3000 RS autosampler, an Acclaim PepMap RSLC analytical column (75 μm by 50 cm, nanoViper, C18, 2 μm, 100 Å; Thermo Fisher Scientific), and an Acclaim PepMap 100 trap column (100 μm by 2 cm, nanoViper, C18, 5 μm, 100 Å; Thermo Fisher Scientific), the tryptic peptides were separated by increasing concentrations of 80% acetonitrile/0.1% formic acid at a flow of 250 nl/min for 158 min and analyzed with a QExactive Plus mass spectrometer (Thermo Fisher Scientific). The instrument was operated in data-dependent acquisition mode to automatically switch between full-scan mass spectrometry (MS) and tandem MS acquisition. Each survey full scan [375 to 1575 mass/charge ratio (m/z)] was acquired with a resolution of 70,000 (at 200 m/z), an AGC (automatic gain control) target of 3 × 10⁶, and a maximum injection time of 54 ms. Dynamic exclusion was set to 15 s. The 12 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the collision cell by higher-energy collisional dissociation (HCD) with a fixed injection time of 54 ms, 17,500 resolution, and an AGC target of 2 × 10⁵.

Adhikari D., Lee I.W., Al-Zubaidi U., Liu J., Zhang Q.H., Yuen W.S., He L., Winstanley Y., Sesaki H., Mann J.R., Robker R.L, & Carroll J. (2022). Depletion of oocyte dynamin-related protein 1 shows maternal-effect abnormalities in embryonic development. Science Advances, 8(24), eabl8070.

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LC–MS/MS Analysis of Peptides

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LC–MS/MS analysis was carried out using the Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to the Easy nLC1200 nano-flow UPLC. Peptides (500 ng of each sample) were injected into the Acclaim PepMap 100 trap column (nanoViper C18, 100 μm × 2 cm, Thermo Scientific) and separated by the Acclaim Pep Map RSLC analytical column (nanoViper C18, 50 μm × 15 cm, Thermo Scientific) set at the flow rate of 300 nL/min. The solvent gradients were set as a linear gradient of 8~38% mobile phase B (80% acetonitrile contains 0.1% formic acid) over 102 min through 120 min run time. The sample was atomized using the nanoESI source. The data-dependent acquisition (Top 20) was carried out using MS survey scans in the 350~1700 m/z range with 70,000 mass resolution. For subsequent MS/MS analysis, the resolution was set to 17,500, and the isolation window was set to 1.6 m/z. The normalized collision energy was 27 eV. Peptides with charge 2~7 were selected.

Liu S., Liu T., Wang E., Cheng Y., Liu T., Chen G., Guo M, & Song B. (2022). Dissecting the Chloroplast Proteome of the Potato (Solanum Tuberosum L.) and Its Comparison with the Tuber Amyloplast Proteome. Plants, 11(15), 1915.

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Mass Spectrometry Proteomics Data Acquisition

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Mass spectrometric data were collected on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, MA, United States) coupled to a an Easy-nLC 1000 system (Thermo Scientific, MA, United States). The tryptic peptides were dissolved in 0.1% formic acid, and 10 μl of peptide sample was loaded onto a 2-cm Acclaim C18 Pepmap trap column, with a flow rate of 10 μl/min for 3 min and subsequently separated on a 75 μm × 15 cm Acclaim Pepmap RSLC analytical column (Thermo Scientific, MA, United States) using a 120 min gradient at a flow rate of 300 nl/min with buffer D (ACN with 0.1% formic acid) ranging from 3% to 32%. The electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used. The fusion mass spectrometer was set at data-dependent mode, allowing the apparatus to automatically switch between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350–1550) were acquired in Orbitrap at a resolution of 120K followed by MS/MS using high-energy collisional dissociation (HCD) at a resolution of 30 K. The AGC target was set to 4 × 10⁵ and the precursor isolation window was 1.6 m/z. MS/MS fixed first mass at 110 m/z. One microscan was recorded using 45-s dynamic exclusion in all cases. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD023576.

Li B., Zhang Y., Qiu D., Francis F, & Wang S. (2021). Comparative Proteomic Analysis of Sweet Orange Petiole Provides Insights Into the Development of Huanglongbing Symptoms. Frontiers in Plant Science, 12, 656997.

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Proteomic Analysis Using QExactive Plus MS

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The proteomic analysis was carried out at Monash Biomedical Proteomics Facility, Monash, Australia. Data-dependent acquisition was performed on a QExactive™ Plus 1 mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to a Dionex UltiMate^® 3000 RSLC nano Liquid Chromatography (LC) system (Thermo Scientific, Waltham, MA, USA) equipped with Acclaim PepMap RSLC analytical column (75 µm × 50 cm, nanoViper, C18, 2 µm, 100Å, Thermo Scientific, Waltham, MA, USA) and Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper, C18, 5 µm, 100Å, Thermo Scientific, Waltham, MA, USA).

Khoo X.H., Paterson I.C., Goh B.H, & Lee W.L. (2019). Cisplatin-Resistance in Oral Squamous Cell Carcinoma: Regulation by Tumor Cell-Derived Extracellular Vesicles. Cancers, 11(8), 1166.

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DDA Mass Spectrometry Protocol for Protein Identification

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Using a Dionex
UltiMate 3000 RSLCnano system equipped with a Dionex UltiMate 3000
RS autosampler, the samples were loaded via an Acclaim PepMap 100
trap column (100 μm × 2 cm, nanoViper, C18, 5 μm,
100 Å; Thermo Scientific) onto an Acclaim PepMap RSLC analytical
column (75 μm × 50 cm, nanoViper, C18, 2 μm, 100
Å; Thermo Scientific). The peptides were separated by elution
with increasing concentrations of 80% ACN/0.1% FA at a flow rate of
250 nL/min for 158 min and analyzed with an Orbitrap Fusion Tribrid
mass spectrometer (Thermo Scientific). Each cycle was set to a fixed
cycle time of 4 s consisting of an Orbitrap full MS1 scan (resolution:
120,000; AGC target: 1 × 10⁶; maximum IT: 54 ms; scan
range: 375–1575 m/z), followed
by several Orbitrap MS2 scans (resolution: 30,000; AGC target: 4 ×
10⁵; maximum IT: 118 ms; isolation window: 1.4 m/z; HCD collision energy: 32%). To minimize
repeated sequencing of the peptides, dynamic exclusion was set to
15 s, and the “exclude isotopes” option was activated.
The raw and analyzed DDA data files have been deposited to the ProteomeXchange
Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner
repository^{23 (link)} with the dataset identifier
PXD017164.

Karthikaichamy A., Beardall J., Coppel R., Noronha S., Bulach D., Schittenhelm R.B, & Srivastava S. (2021). Data-Independent-Acquisition-Based Proteomic Approach towards Understanding the Acclimation Strategy of Oleaginous Microalga Microchloropsis gaditana CCMP526 in Hypersaline Conditions. ACS Omega, 6(34), 22151-22164.

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Quantitative Proteomics and Phosphoproteomics

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Six tissue samples were transferred into a low protein binding tube and lysed with PMSF buffer to extract protein. Protein concentration was determined by BCA assay. After tryptic digestion and TMT labeling, the TMT-labeled peptide mix was fractionated using an Agilent Zorbax Extend C18 column on Agilent 1100 HPLC. Protein samples were separated on the Acclaim PepMap RSLC analytical column (RP-C18, Thermo Fisher Scientific, USA). Shotgun proteomics analyses were performed using Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Protein with at least one unique peptide was identified at a false discovery rate (FDR) less than 1.0% on peptide or protein level. For phosphoproteomic samples, phosphopeptides were enriched using titanium dioxide beads (TiO2), and LC-MS/MS analysis was similar to proteomic experiment. The complete experimental and bioinformatics process can be seen in the supporting materials.

Jiang B., Yang J., He R., Wang D., Huang Y., Zhao G., Ning M., Zeng T, & Li G. (2023). Integrated multi-omics analysis for lung adenocarcinoma in Xuanwei, China. Aging (Albany NY), 15(23), 14263-14291.

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Comprehensive Proteome Profiling with DIA-MS

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Samples were analyzed on an UltiMate 3000 RSLC nano LC system (Thermo Fisher Scientific) coupled to an LTQ-Orbitrap mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific). Peptides were loaded via an Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper, C18, 5 μm, 100 Å, Thermo Fisher Scientific) and subsequent peptide separation was on an Acclaim PepMap RSLC analytical column (75 μm × 50 cm, nanoViper, C18, 2 μm, 100 Å, Thermo Fisher Scientific). For each liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, 1 μg of peptides as measured by a nanodrop 1000 spectrophotometer (Thermo Fisher Scientific) was loaded on the pre-column with microliter pickup. Peptides were eluted using a 2 h linear gradient of 80% ACN/0.1% FA flowing at 250 nL/min using a mobile phase gradient of 2.5–42.5% ACN. The eluting peptides were interrogated with an Orbitrap mass spectrometer. The HRM DIA method consisted of a survey scan (MS1) at 35,000 resolution (automatic gain control target 5^e6 and maximum injection time of 120 ms) from 400 to 1,220 m/z followed by tandem MS/MS scans (MS2) through 19 overlapping DIA windows increasing from 30 to 222 Da. MS/MS scans were acquired at 35,000 resolution (automatic gain control target 3^e6 and auto for injection time). Stepped collision energy was 22.5%, 25%, 27.5%, and a 30 m/z isolation window. The spectra were recorded in profile type.

Nguyen E.V., Pereira B.A., Lawrence M.G., Ma X., Rebello R.J., Chan H., Niranjan B., Wu Y., Ellem S., Guan X., Wu J., Skhinas J.N., Cox T.R., Risbridger G.P., Taylor R.A., Lister N.L, & Daly R.J. (2019). Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment. Molecular & Cellular Proteomics : MCP, 18(7), 1410-1427.

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Quantitative LC-MS/MS Proteomics Analysis

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All samples were analyzed on an LC-ESI-MS/MS consisting of an EASY-nLC system (Thermo Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Scientific) with a Nanospray FLex ion source (Thermo Scientific). Peptides were loaded on a reverse phase Acclaim PEPMAP NANOTRAP column (C18, 100 Å, 100 μm. × 2 cm, (Thermo Scientific)) in solvent A (0.1% FA) followed by separation on a reverse phase ACCLAIM PEPMAP RSLC analytical column (C18, 100 Å, 75 um × 50 cm (Thermo Scientific)). Peptides were eluted by constant flow at 300 nL/min during a 60 min ramped gradient from 5 to 100% of solvent B (0.1% FA in 80% ACN, Fischer Scientific). MS was operated in positive ion and data dependent top-20 mode, were the (up to) 20 most intense MS1 precursors were selected for higher energy C-trap dissociation (HCD) fragmentation at 28 eV using a window of isolation of 1.2 m/z. Survey scans were obtained at a resolution of 60.000 at 200 m/z and HCD spectra were obtained at 15.000 at 200 m/z. Maximum ion injection time was set to 50 for MS and 45 for MS/MS scans. The underfill ratio was set to 3.5% and a dynamic exclusion of 30 sec was applied. During acquisition, “peptide match” and “exclude isotopes” were enabled.

Gregersen S., Kongsted A.S., Nielsen R.B., Hansen S.S., Lau F.A., Rasmussen J.B., Holdt S.L, & Jacobsen C. (2021). Enzymatic extraction improves intracellular protein recovery from the industrial carrageenan seaweed Eucheuma denticulatum revealed by quantitative, subcellular protein profiling: A high potential source of functional food ingredients. Food Chemistry: X, 12, 100137.

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LC-MS/MS Analysis of Peptides

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LC-MS/MS analysis was performed as previously described (Matsushima et al., 2021 ). Briefly, approximately 1 g of peptide was separated with the Easy-nLC1000 system (ThermoScientific) using Acclaim PepMap100 trap column (20 × 0.075mm, 3um, Thermo Scientific) and the Acclaim PepMap RSLC analytical column (150 × 0.05mm, 2um, Thermo Scientific) and analyzed on Q-Exactive Orbitrap mass analyzer (Thermo Scientific). Data analysis was performed using Proteome Discoverer software (Thermo Scientific) for protein identification through SequestHT algorithm against human protein Uniprot database.

Fujii M., Setoyama D., Gotoh K., Dozono Y., Yagi M., Ikeda M., Ide T., Uchiumi T, & Kang D. (2022). TFAM expression in brown adipocytes confers obesity resistance by secreting extracellular vesicles that promote self-activation. iScience, 25(9), 104889.

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Peptide Identification for Proteomics and Immunopeptidomics

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For identification of peptides for both proteomics and immunopeptidomics we used Data dependent acquisition (DDA) approach. Using a Dionex UltiMate 3000 RSLCnano system equipped with a Dionex UltiMate 3000 RS autosampler, the samples were loaded via an Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper, C18, 5 µm, 100Å; Thermo Scientific) onto an Acclaim PepMap RSLC analytical column (75 µm × 50 cm, nanoViper, C18, 2 µm, 100Å; Thermo Scientific). The peptides were separated by increasing concentrations of 80% ACN/0.1% FA at a flow of 250 nl/min for 65 min and analyzed with a QExactive Plus mass spectrometer (Thermo Scientific). In each cycle, a full ms1 scan (resolution: 70.000; AGC target: 3e6; maximum IT: 120 ms; scan range: 375–1800 m/z) preceded up to 12 subsequent ms2 scans (resolution: 17.500; AGC target: 1e5; maximum IT: 120 ms; isolation window: 1.8 m/z; scan range: 200–2,000 m/z; NCE: 27).
For quantitative proteomics we used data independent acquisition (DIA) approach the identical LC-MS/MS instrument setup above was used. Twenty-five sequential DIA windows with an isolation width of 24 m/z between 375 and 975 m/z were acquired. A 158-min gradient of increasing concentrations of 80% ACN/0.1% FA was used to separate the peptides for DIA acquisition.

Goncalves G., Mullan K.A., Duscharla D., Ayala R., Croft N.P., Faridi P, & Purcell A.W. (2021). IFNγ Modulates the Immunopeptidome of Triple Negative Breast Cancer Cells by Enhancing and Diversifying Antigen Processing and Presentation. Frontiers in Immunology, 12, 645770.

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