The TMT labeled samples were fractionated by offline basic pH reverse phase LC, yielding 40 fractions. Each fraction was analyzed by the acidic pH reverse phase LC-MS/MS [26 (link)]. In the acidic pH LC-MS/MS analysis, each fraction was run sequentially on a column (75 μm × 20 cm for the whole proteome, 50 μm × ∼30 cm for phosphoproteome, 1.9 μm C18 resin, 65 °C to reduce backpressure) interfaced with a Q Exactive HF Orbitrap or Fusion MS (Thermo Fisher). Peptides were eluted by a 2–3 h gradient (buffer A: 0.2% formic acid, 5% DMSO; buffer B: buffer A plus 65% acetonitrile). MS settings included the MS1 scan (410–1600 m/z, 60,000 or 120,000 resolution, 1 × 106 AGC and 50 ms maximal ion time) and 20 data-dependent MS2 scans (fixed first mass of 120 m/z, 60,000 resolution, 1 × 105 AGC, 100–150 ms maximal ion time, HCD, 35–38% normalized collision energy, ∼1.0 m/z isolation window).
Q exactive hf orbitrap or fusion ms
Manufactured by Thermo Fisher Scientific
The Q Exactive HF Orbitrap or Fusion MS is a high-resolution mass spectrometer designed for advanced proteomics and metabolomics analysis. It utilizes Orbitrap mass analyzer technology to provide accurate mass measurements and high-resolution data. The instrument is capable of performing both full-scan and data-dependent acquisition modes to enable comprehensive characterization of complex samples.
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Lab products found in correlation
2 protocols using q exactive hf orbitrap or fusion ms
1
Offline RP-LC-MS/MS for Proteome and Phosphoproteome
2
Offline Basic pH Reverse Phase LC-MS/MS
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The TMT labeled samples were fractionated by o ine basic pH reverse phase LC, yielding 40 fractions. Each fraction was analyzed by the acidic pH reverse phase LC-MS/MS (Wang, 2015, Journal of Proteome Research). In the acidic pH LC-MS/MS analysis, each fraction was run sequentially on a column (75 µm x 20 cm for the whole proteome, 50 µm x ∼30 cm for phosphoproteome, 1.9 µm C18 resin from Dr. Maisch GmbH, 65°C to reduce backpressure) interfaced with a Q Exactive HF Orbitrap or Fusion MS (Thermo Fisher). Peptides were eluted by a 2-3 hr gradient (buffer A: 0.2% formic acid, 5% DMSO; buffer B: buffer A plus 65% acetonitrile). MS settings included the MS1 scan (410-1600 m/z, 60,000 or 120,000 resolution, 1 × 10 6 AGC and 50 ms maximal ion time) and 20 data-dependent MS2 scans ( xed rst mass of 120 m/z, 60,000 resolution, 1 × 10 5 AGC, 100-150 ms maximal ion time, HCD, 35-38% normalized collision energy, ∼1.0 m/z isolation window).
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