Nano-LC-MS/MS Peptide Identification

Peptide material from pull-down experiments was resuspended in 20 μl water with 0.1% TFA and 2% acetonitrile. Samples were then injected into a Dionex Ultimate 3000 nano-ultra LC system (Thermo Fisher) coupled on-line to a Q Exactive mass spectrometer (Thermo Scientific) as described previously (27 (link), 28 (link)). In brief, the samples were separated on an EASY-Spray LC column (PepMap RSLC C18, 500 mm × 75 μm ID, 2 μm particle size, Thermo Scientific) over 60 min for GlyGly immunoprecipitation and 120 min for the total proteome (gradient of 2–35% acetonitrile in 5% DMSO and 0.1% formic acid) at 250 nl/min. Acquisition of MS1 scans was performed at a resolution of 60,000 at 200 m/z. The most abundant precursor ions (top 12) were selected for HCD fragmentation.

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Kaokhum N., Pinto-Fernández A., Wilkinson M., Kessler B.M, & Ismail H.M. (2022). The Mechano-Ubiquitinome of Articular Cartilage: Differential Ubiquitination and Activation of a Group of ER-Associated DUBs and ER Stress Regulators. Molecular & Cellular Proteomics : MCP, 21(12), 100419.

Publication 2022

Dionex Ultimate 3000 Q Exactive mass spectrometer EASY-Spray LC column

Acetonitrile Dmso Formic acid Immunoprecipitation Ions Peptide Proteome Pull Scans

Corresponding Organization : University of Sheffield

Other organizations : University of Oxford

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Variable analysis

independent variables

Resuspension in 20 μl water with 0.1% TFA and 2% acetonitrile
Separation on an EASY-Spray LC column (PepMap RSLC C18, 500 mm × 75 μm ID, 2 μm particle size, Thermo Scientific) over 60 min for GlyGly immunoprecipitation and 120 min for the total proteome
Gradient of 2–35% acetonitrile in 5% DMSO and 0.1% formic acid at 250 nl/min

dependent variables

Acquisition of MS1 scans at a resolution of 60,000 at 200 m/z
Selection of the most abundant precursor ions (top 12) for HCD fragmentation

control variables

Coupling of the Dionex Ultimate 3000 nano-ultra LC system (Thermo Fisher) to the Q Exactive mass spectrometer (Thermo Scientific)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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