Quantitative LC-MS/MS Proteomics Protocol

The 24 fractions were analyzed by LC/MS/MS using an Ultimate 3000 nLC coupled to an Orbitrap Exploris480 mass spectrometer (Thermo Scientific) using a turboTMT method.^{33 (link)} Samples were injected onto an Ion Opticks Aurora C18 column (75 μm id × 15 cm, 1.6 μm particle size) and separated over a 70 min method. The gradient for separation consisted of 5−42% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. The Exploris480 was operated in turboTMT mode with a cycle time of 3 s. Resolution for the precursor scan (m/z 375−1400) was set to 60 000 with a AGC target set to standard and a maximum injection time set to auto. MS2 scans (30 000 resolution) consisted of higher collision dissociate (HCD) set to 38, AGC target set to 300%, maximum injection time set to auto, isolation window of 0.7 Da, and fixed first mass of 110 m/z.

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Hanley R.P., Nie D.Y., Tabor J.R., Li F., Sobh A., Xu C., Barker N.K., Dilworth D., Hajian T., Gibson E., Szewczyk M.M., Brown P.J., Barsyte-Lovejoy D., Herring L.E., Wang G.G., Licht J.D., Vedadi M., Arrowsmith C.H, & James L.I. (2023). Discovery of a Potent and Selective Targeted NSD2 Degrader for the Reduction of H3K36me2. Journal of the American Chemical Society, 145(14), 8176-8188.

Publication 2023

Ultimate 3000 nLC Orbitrap Exploris480

Corresponding Organization : Structural Genomics Consortium

Other organizations : University of Florida Health

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Measured outcomes from the LC/MS/MS analysis

control variables

Flow rate of 250 nl/min
Mobile phase A (0.1% formic acid in water)
Mobile phase B (0.1% formic acid in 80% ACN)
Gradient separation of 5-42% mobile phase B
Ion Opticks Aurora C18 column (75 μm id × 15 cm, 1.6 μm particle size)
Precursor scan range of m/z 375-1400
Precursor scan resolution of 60,000
MS2 scan resolution of 30,000
HCD fragmentation set to 38
Isolation window of 0.7 Da
Fixed first mass of 110 m/z

controls

No positive or negative controls were explicitly mentioned in the provided information.

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