LC-MS/MS Proteomic Analysis Protocol

Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was performed in biological quadruplicate using a Dionex Ultimate 3000 nano-ultra high pressure reverse phase chromatography coupled on-line to a Q Exactive High Field (HF) mass spectrometer (Thermo Scientific) as described^{40 (link)}. Samples were separated on an EASY-Spray PepMap RSLC C18 column (500 mm × 75 µm, 2 µm particle size, Thermo Scientific) over a 60 min gradient of 2-35% acetonitrile in 5% DMSO, 0.1% formic acid at 250 nl/min. MS1 scans were acquired at a resolution of 60,000 at 200 m/z and the top 12 most abundant precursor ions were selected for HCD fragmentation.

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Stangl A., Elliott P.R., Pinto-Fernandez A., Bonham S., Harrison L., Schaub A., Kutzner K., Keusekotten K., Pfluger P.T., El Oualid F., Kessler B.M., Komander D, & Krappmann D. (2019). Regulation of the endosomal SNX27-retromer by OTULIN. Nature Communications, 10, 4320.

Publication 2019

Ultimate 3000 Q Exactive High Field (HF) mass spectrometer EASY-Spray PepMap RSLC C18 column

Acetonitrile Biological Dmso Formic acid Ions Liquid chromatography Mass spectrometry mass spectrometry Pressure Reverse phase chromatography Scans

Corresponding Organization : Walter and Eliza Hall Institute of Medical Research

Other organizations : Helmholtz Zentrum München, University of Oxford, Heinrich Heine University Düsseldorf, Deutsches Diabetes-Zentrum e.V., German Center for Diabetes Research

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Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis

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Dionex Ultimate 3000 nano-ultra high pressure reverse phase chromatography
Q Exactive High Field (HF) mass spectrometer (Thermo Scientific)
EASY-Spray PepMap RSLC C18 column (500 mm × 75 µm, 2 µm particle size, Thermo Scientific)
60 min gradient of 2-35% acetonitrile in 5% DMSO, 0.1% formic acid at 250 nl/min
MS1 scans at a resolution of 60,000 at 200 m/z
Top 12 most abundant precursor ions selected for HCD fragmentation

controls

No positive or negative controls were explicitly mentioned.

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