Tryptic peptides were separated on a nano-ACQUITY UPLC analytical column (BEH130 C18, 1.7 μm, 75 μm × 200 mm, Waters) over a 165-min linear acetonitrile gradient (3%–40%) with 0.1% formic acid on a Waters nano-ACQUITY UPLC system and analyzed on a coupled Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer as described previously.45 (link) Full scans were acquired at a resolution of 120 000 with an automatic gain control (AGC) target value of 106 and a maximum injection time of 50 ms. Precursors were selected for fragmentation by higher-energy collisional dissociation at a normalized collision energy of 32% for a maximum 3-s cycle. Products were analyzed in orbitrap at a resolution of 15 000 with an AGC target value of 103 or in ion trap with an AGC target value of 104 in parallel within a maximum injection time of 246 ms by applying an abundance dependent decision tree logic. Interrogated ions were dynamically excluded from reselection for 60 s.
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Nano-UPLC-MS/MS Tryptic Peptide Analysis
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Corresponding Organization : University of Maryland, Baltimore
Other organizations : NOSM University
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Variable analysis
independent variables
- Nano-ACQUITY UPLC analytical column (BEH130 C18, 1.7 μm, 75 μm × 200 mm, Waters)
- Linear acetonitrile gradient (3%–40%) with 0.1% formic acid
- Fragmentation by higher-energy collisional dissociation at a normalized collision energy of 32%
- Separation of tryptic peptides
- Mass spectrometry analysis on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer
- Full scans acquired at a resolution of 120,000
- Precursors selected for fragmentation
- Products analyzed in orbitrap at a resolution of 15,000 or in ion trap
- Waters nano-ACQUITY UPLC system
- Automatic gain control (AGC) target values
- Maximum injection times
- Dynamic exclusion of interrogated ions for 60 s
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