Cells were harvested with intra-cellular proteins extracted and digested with trypsin. Peptides were then labeled with TMT using a TMT10plex mass tag labeling kit (Thermo Fisher Scientific) per the manufacturer’s instructions. TMT-labeled peptides were then separated by high pH reverse-phase high performance liquid chromatography (HPLC) with C18 columns (Agilent BioTek) and dried in a vacuum centrifuge. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was subsequently performed. All fragments were sequentially dissolved in aqueous solution (0.1% formic acid and 2% acetonitrile), loaded onto a home-made reverse-phase analytical column, and subjected to an EASY-nLC™ 1000 ultraperformance liquid chromatography (UPLC) system (Thermo Fisher Scientific) at a constant flow rate of 400 nL/min. For MS settings, the applied electrospray voltage was 2.0 kV and the m/z range was 350 to 1800 for the complete scan. Peptide fragments were quantified with Parallel Reaction Monitoring (PRM). Proteins were identified by Swissprot database and quantified using Proteome Discoverer 2.0.
Easy nlc 1000 ultraperformance liquid chromatography
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EASY-nLC™ 1000 is an ultraperformance liquid chromatography system designed for high-sensitivity analysis of small sample volumes. It features a robust and reliable design to deliver consistent and reproducible separations.
Automatically generated - may contain errors
2 protocols using easy nlc 1000 ultraperformance liquid chromatography
1
Quantitative Proteomics of Cells
2
Fractionated Peptide Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fractionated peptides were dissolved in aqueous solution with 0.1% formic acid and 5% acetonitrile, and loaded onto a Thermo Scientific EASY trap column (100 mm × 2 cm, 5 mm, 100 Å, C18, Thermo Fisher Scientific) and an analytical column (75 mm × 25 cm, 5 mm, 100 Å, C18, Thermo Fisher Scientific). Then, the peptides were separated for 90 min with a gradient of 5% to 28% Solvent B (0.1% formic acid, 98% acetonitrile) for 40 min followed by 28%–90% Solvent B for 2 min and then 90% Solvent B for 18 min, at a flow rate of 600 nL/min on an EASY-nLC 1000 Ultra-performance liquid chromatography (UPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). After liquid-phase separation, the peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) coupled online to the HPLC.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!