For mass spectrometry, samples were run short-distance on a 4–12% SDS-PAGE gel and stained with Coomassie Blue. The lane was excised from the gel after which proteins were reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested with trypsin (mass spec grade, Promega) overnight at 37 °C and peptides were extracted with acetonitrile. Digests were dried in a vacuum centrifuge and reconstituted in 10% formic acid for MS analysis. Peptide mixtures (10% of total digest) were loaded directly on the analytical column and analyzed by nanoLC-MS/MS on an Orbitrap Fusion Tribrid mass spectrometer equipped with a Proxeon nLC1000 system (Thermo Scientific).
Proxeon nlc1000 system
Manufactured by Thermo Fisher Scientific
The Proxeon nLC1000 system is a liquid chromatography instrument designed for nanoflow separations. It features a modular design, supports flow rates from 20 nL/min to 2 μL/min, and is compatible with various column configurations. The system is intended for use in applications requiring high-resolution separation of complex samples, such as those encountered in proteomics research.
Automatically generated - may contain errors
3 protocols using proxeon nlc1000 system
1
Protein Identification via Mass Spectrometry
2
Tubulin Proteomics by Tryptic Digestion
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Tubulin bands were excised from the coomassie stained gel, after which proteins were reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested with trypsin (mass spec grade, Promega) overnight at 37°C and peptides were extracted with acetonitrile. Digests were dried in a vacuum centrifuge and reconstituted in 10% formic acid for MS analysis. Peptide mixtures (10% of total digest) were loaded directly on the analytical column and analyzed by nanoLC-MS/MS on an Orbitrap Fusion Tribrid mass spectrometer equipped with a Proxeon nLC1000 system (Thermo Scientific) as described previously [39 (link)]. Solvent A was 0.1% formic acid/water and solvent B was 0.1% formic acid/80% acetonitrile. Peptides were eluted from the analytical column at a constant flow of 250 nl/min in a 90-min gradient, containing a 74-min linear increase from 5% to 24% solvent B, followed by a 16-min wash at 80% solvent B.
3
In-Gel Proteome Profiling by Mass Spectrometry
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Immunoprecipitation beads were heated in sample loading buffer for 5 minutes. at 95 C, eluates were run into the stacking of a 4% to 12% Bis-Tris gel and coomassie-stained bands were excised. Proteins were reduced with 6.5 mmol/L DTT, alkylated with 54 mmol/L iodoacetamide and digested in-gel with trypsin (Gold, mass spectrometry grade, Promega, 3 ng/mL) overnight at 37 C. Extracted peptides were vacuum dried, reconstituded in 10% formic acid and analyzed by nanoLC-MS/MS on an Orbitrap Fusion Tribrid mass spectrometer equipped with a Proxeon nLC1000 system (Thermo Scientific). Peptides were loaded direct-ly on the analytical column (Agilent Poroshell EC-C18 120 2.7 mm, 50 mm  500 mm, packed in-house) and separated in a 140-minutes gradient containing a 124-minutes linear increase from 6% to 30% solvent B (0.1% formic acid/80% acetonitrile), with 0.1% formic acid/water as Solvent A. Further settings were as described previously (25) .
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