Liquid chromatography separation of a 2 μg sample was achieved by reversed-phase chromatography using a NanoLC Ultra 2D HPLC (Eksigent) equipped with a nanoflex cHiPLC set to 37°C. A 90-minute gradient was used for peptide separation, as described (7 (link)), followed by elution directly into a 5500 QTrap (AbSciex). Peptide precursors were selected in quadrupole 1 (Q1), fragmented in q2, and the top 3–4 transitions were selected for monitoring in Q3. All Q1 and Q3 masses were measured at unit resolution. A 7-minute scheduling window was applied with a 1.5-second cycle time. Method development and peak analysis were done using Skyline software (18 (link)).
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Targeted Peptide Quantification by LC-MS/MS
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Corresponding Organization : University of Wisconsin–Madison
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Variable analysis
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- Liquid chromatography separation method
- Reversed-phase chromatography
- NanoLC Ultra 2D HPLC (Eksigent)
- Nanoflex cHiPLC set to 37°C
- 90-minute gradient for peptide separation
- Elution into a 5500 QTrap (AbSciex)
- Peptide precursor selection in quadrupole 1 (Q1)
- Peptide fragmentation in q2
- Selection of top 3-4 transitions for monitoring in Q3
- Q1 and Q3 masses measured at unit resolution
- 7-minute scheduling window with 1.5-second cycle time
- Liquid chromatography separation of a 2 μg sample
- Temperature set to 37°C for nanoflex cHiPLC
- No positive or negative controls were explicitly mentioned in the provided information.
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