Following reconstitution in 0.1% formic acid, samples were loaded on a 75 μm × 15 cm fused silica capillary (custom‐made) packed with C18Q resin (Reprosil‐PUR 120, 1.9 μm, Dr. Maisch) and separated using EASY nLC 1,200 liquid chromatography (Thermo Scientific). For the C9orf72 interactome, peptides were separated using a 35 min ACN gradient in 0.1% formic acid at a flowrate of 400 nl/min (10–38% ACN for 23 min, 38–60% ACN for 2 min and 60–100% ACN for 5 min). For the C9orf72 ubiquitination status, peptide separation was performed with a 60 min ACN gradient in 0.1% formic acid at a flowrate of 400 nl/min (10–38% ACN for 35 min, 38–60% ACN for 5 min and 60–100% ACN for 10 min). Separated peptides were detected using Q Exactive HF mass spectrometer (Thermo Scientific).
Easy nlc 1200 liquid
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The EASY-nLC 1200 liquid chromatography system is a high-performance nanoflow HPLC designed for sensitive and high-resolution separation of complex samples. It features a dual-pump design, precise flow control, and compatibility with various column formats to support a wide range of application needs.
Automatically generated - may contain errors
Lab products found in correlation
Reprosil pur c18, by Dr. Maisch (2 mentions)
Q exactive hf, by Thermo Fisher Scientific (2 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (1 mentions)
Accucore 150 resin, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap 100 c18 trap column, by Thermo Fisher Scientific (1 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap 100 c18, by Thermo Fisher Scientific (1 mentions)
6 protocols using easy nlc 1200 liquid
1
High-throughput Proteomics Workflow for C9orf72 Interactome and Ubiquitination Analysis
2
Proteome Analysis by Orbitrap Fusion Lumos
3
Proteomic Analysis of Exosomes
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The proteomic analysis of exosomes was performed using a label-free analysis technology. Protein was extracted from exosomes, quantified with BCA assay, digested by trypsin, and dried by centrifugal concentration. Then, the peptides were desalted using MonoSpin C18 desalting column (GL Sciences Inc., Japan, 5010-21701), analyzed by Orbitrap Fusion Lumos tandem mass spectrometry (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 liquid chromatography system (Thermo Fisher Scientific). Protein identification and quantification were performed using Proteome Discoverer 2.4 (Database: Swissprot; taxonomy: Homo sapiens) with the default setting. The experiments and data analysis were offered by Wayen Biotechnologies (Shanghai, China). We used the Metascape platform online analytical tool to further analyze part of the proteomic analysis data.37 (link) The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE38 (link) partner repository with the dataset identifier PXD033839.
4
Mass Spectrometry-based Proteomic Analysis
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The digested and desalted samples were analyzed using a QExactive HF mass spectrometer interfaced with an Easy-nLC 1200 liquid chromatography system (both Thermo Fisher Scientific). Peptides were trapped on an Acclaim PepMap 100 C18 trap column (100 μm by 2 cm; particle size, 5 μm; Thermo Fischer Scientific) and separated on an in-house packed analytical column (75 μm by 300 mm; particle size, 3 μm; Reprosil-Pur C18, Dr. Maisch). A stepped gradient used was from 7 to 35% solvent B in 97 min, followed by an increase to 48% in 8 min and to 100% solvent B in 5 min at a flowrate of 300 nl/min. Solvent A was 0.2% formic acid, and solvent B was 80% acetonitrile in 0.2% formic acid. The mass spectrometer was operated in data-dependent acquisition (DDA) mode where the MS1 scans were acquired at a resolution of 60,000 and a scan range from 400 to 1600 mass/charge ratio (m/z). The 10 most intense ions with a charge state of 2 to 4 were isolated with an isolation window of 1.2 m/z and fragmented using normalized collision energy of 28. The MS2 scans were acquired at a resolution of 30,000, and the dynamic exclusion time was set to 20 s.
5
Mass Spectrometry-based Proteomics Workflow
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After sample processing, mass spectrometry data were collected using a Q Exactive Plus mass spectrometer (Thermo Scientific) in series with an EASY-nLC 1200 liquid phase LC/MS system (Thermo Scientific). The mass spectral data were searched with MaxQuant (V1.6.6) software, and the database search algorithm Andromeda was used to search the Human's Proteome Reference Database in UniProt (2020-05-10, containing 75074 protein sequences).
6
High-throughput Proteomics Analysis Pipeline
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The digested and desalted samples were analysed using a QExactive HF mass spectrometer interfaced with an Easy-nLC 1200 liquid chromatography system (both Thermo Fisher Scientific). Peptides were trapped on an Acclaim PepMap 100 C18 trap column (100 μm x 2 cm, particle size 5 μm, Thermo Fischer Scientific) and separated on an in-house packed analytical column (75 μm x 300 mm, particle size 3 μm, Reprosil-Pur C18, Dr. Maisch). A stepped gradient used was from 7% to 35% solvent B in 97 min followed by an increase to 48% in 8 min and to 100% solvent B in 5 min at a flowrate of 300 nL/min. Solvent A was 0.2% formic acid and solvent B was 80% acetonitrile in 0.2% formic acid. The mass spectrometer was operated in data-dependent mode (DDA) where the MS1 scans were acquired at a resolution of 60000 and a scan range from 400 to 1600 m/z. The 10 most intense ions with a charge state of 2 to 4 were isolated with an isolation window of 1.2 m/z and fragmented using normalized collision energy of 28. The MS2 scans were acquired at a resolution of 30000 and the dynamic exclusion time was set to 20 s.
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