Yeast cells were grown in 1.2 liters of YPD media until an OD600 of 1.0, washed with PBS, and flash frozen in liquid nitrogen. Thawed cell pellets were resuspended in IP buffer [40 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 10% glycerol, and 0.1% Tween-20] and lysed with glass beads using a Biospec bead beater. The lysate was centrifuged at 45,000 rpm at 4°C for 1.5 hours and the supernatant was incubated with anti-FLAG M2 affinity gel (GenScript) at 4°C for 4 hours. The beads were washed extensively in IP buffer. The immunoprecipitated proteins were digested overnight with sequencing-grade trypsin (Promega) at 37°C and the supernatant peptides were then desalted using C18 columns (Thermo Fisher Scientific) and lyophilized. The dried peptides were reconstituted in 0.1% FA and loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher Scientific) using a Thermo Easy nLC 1000 LC system (Thermo Fisher Scientific) connected to Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The raw mass spectrometry data were searched against the S. cerevisiae proteome database from Uniport (https://uniprot.org/proteomes/UP000002311 ) using Sequest HT, MS Amanda, and ptmRS algorithms in Proteome Discoverer 2.3 (Thermo Fisher Scientific).
Thermo easy nlc 1000 lc system
Manufactured by Thermo Fisher Scientific
The Thermo Easy nLC 1000 LC system is a liquid chromatography system designed for high-performance liquid chromatography (HPLC) and nano-flow liquid chromatography (nanoLC) applications. The system features a high-pressure gradient pump, an autosampler, and a temperature-controlled column compartment. It is capable of delivering precise and reproducible flow rates for analytical and preparative separations.
Automatically generated - may contain errors
Lab products found in correlation
Acclaim pepmap 100 c18 lc column, by Thermo Fisher Scientific (4 mentions)
Sequencing grade trypsin, by Promega (3 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Bead beater, by Biospec (2 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 affinity gel, by GenScript (1 mentions)
C18 column, by Thermo Fisher Scientific (1 mentions)
Easy spray c18 column, by Thermo Fisher Scientific (1 mentions)
Silica nano electrospray emitter, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using thermo easy nlc 1000 lc system
1
Yeast Proteome Immunoprecipitation and Mass Spectrometry
2
Disulfide Reduction, Alkylation, and Tryptic Digestion
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Proteins were disulphide-reduced by 25 mM DTT at 37 °C for 40 min. Cysteines were alkylated by 50 mM iodoacetamide. Proteins were digested with sequencing-grade trypsin (Promega) at 37 °C overnight and the supernatant was desalted using C18 solid-phase cartridges and lyophilized. The dried peptides were reconstituted in 0.1% FA and loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher) utilizing a Thermo Easy nLC 1000 LC system (Thermo Fisher) connected to Q Exactive HF mass spectrometer (Thermo Fisher) and analyzed66 (link).
3
Affinity Purification and Mass Spectrometry of FLAG-Tagged Proteins
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Yeast cells were grown in 1.2 L YPD media until an OD600 of 1.0, washed with PBS and flash frozen in liquid nitrogen. Thawed cell pellets were resuspended in IP buffer (40 mM HEPES pH7.5, 150 mM NaCl, 10% glycerol, 0.1% Tween-20) and lysed with glass beads using a Biospec bead beater. The lysate was centrifuged at 45,000 rpm at 4 °C for 1.5 h and the supernatant was incubated with anti-FLAG agarose beads at 4 °C for 4 h. The beads were washed extensively in IP buffer. Proteins were disulphide-reduced by 25 mM DTT at 37 °C for 40 min, alkylated by adding 50 mM iodoacetamide, and then digested with sequencing-grade trypsin (Promega) at 37 °C overnight. The supernatant was desalted using C18 solid-phase cartridges and lyophilized. The dried peptides were reconstituted in 0.1% FA and loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher) utilizing a Thermo Easy nLC 1000 LC system (Thermo Fisher) connected to Q Exactive HF mass spectrometer (Thermo Fisher) and data were analyzed as described68 (link).
4
Nano-LC-MS/MS Peptide Analysis Protocol
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Peptides were analysed by on-line nanoflow liquid chromatography (LC) using the Thermo EASY-nLC 1000 LC system (Thermo Fisher Scientific) coupled with Q-Exactive mass spectrometer (Thermo Fisher Scientific). Samples were loaded onto an Easy-Spray C18 column (50 cm, inner diameter 75 µm), fused to a silica nano-electrospray emitter (Thermo Fisher Scientific). Chromatography was performed at 35°C with a buffer system consisting of 0.1% formic acid (buffer A) and 80% acetonitrile in 0.1% formic acid (buffer B). The peptides were separated over a 97 min linear gradient of 3.8-50% buffer B at a flow rate of 300 nl/min. The Q-Exactive was operated in data-dependent mode with dynamic exclusion and survey scans acquired at a resolution of 70,000. The 10 most abundant isotope patterns with charge states +2, +3 and/or +4 from the survey scan were selected with an isolation window of 2.0 Th and fragmented by higher energy collisional dissociation with normalised collision energies of 30. The maximum ion injection times for the survey scan and the MS/MS scans were 250 and 100 ms, respectively, and the ion target value was set to 1E6 for survey scans and 1E4 for the MS/MS scans.
5
Quantitative Proteomics Analysis Workflow
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Peptides were analyzed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The desalted tryptic peptide was loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher Scientific) utilizing a Thermo Easy nLC 1000 LC system (Thermo Fisher Scientific) connected to the Q Exactive mass spectrometer. The peptides were eluted with a 5–48% gradient of acetonitrile with 0.1% formic acid over 55 min with a flow rate of 300 nL min−1. The raw MS data were collected and analyzed in Proteome Discoverer 2.2 (Thermo Fisher Scientific) with Sequest HT software and was searched against the Human Proteome database.
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