TITLE: Untargeted Top-down Proteomics by CZE-MS/MS on Eclipse
AUTHORS: Kevin  Jooß, Bryon Drown, Rafael Melani, Kelleher Research Group

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Describes the CZE-MS/MS data acquisition procedure for top-down proteomics samples using the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer</div></div>

[STEPS]
?. Samples were analyzed on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer in line with a Sciex CESI 8000 Plus
?. Samples were prepared in ( ) 0.3% acetic acid and transferred to sample vials.
10 µl
?. Prior each sample injection, the capillary is flushed with 0.1 M HCl, filled with new background electrolyte, and conductive liquid was replaced. Sample is injected hydrodynamically at 2.5 psi for 60 sec (estimated 20 nL injection volume). Separation is performed at 15kV with 0.5 psi supplemental pressure for 60 minutes.Method for CESI 8000 Plus is summarized below:  Time [min]    Event    Value    Duration    Inlet    Outlet    Summary         Rinse - Pressure    100 psi    5.00 min    BI: C1    BO: A1    forward         Rinse - Pressure    100 psi    3.00 min    BI: A1    BO: A1    reverse         Rinse - Pressure    100 psi    5.00 min    BI: A1    BO: A1    forward         Inject - Pressure    2.5 psi    60 sec    sample vial    BO: A1    forward         Wait         0.00 min    BI: D1    BO: A1    dipping         Inject - Pressure    2.5 psi    10 sec    BI: B1    BO: A1    forward    0.00    Separation - Voltage    15 kV  0.5 psi    60.00 min    BI: B1    BO: A1    1.0 min ramp, normal
  polarity, both    1.00    Relay On                             60.00    Separation - Voltage    1.0 kV&nbsp;   5 psi    5.00 min    BI: B1    BO: A1    5.0 min ramp, normal
  polarity, both    65.00    End
Time [min]    Event    Value    Duration    Inlet    Outlet    Summary         Rinse - Pressure    100 psi    5.00 min    BI: C1    BO: A1    forward         Rinse - Pressure    100 psi    3.00 min    BI: A1    BO: A1    reverse         Rinse - Pressure    100 psi    5.00 min    BI: A1    BO: A1    forward         Inject - Pressure    2.5 psi    60 sec    sample vial    BO: A1    forward         Wait         0.00 min    BI: D1    BO: A1    dipping         Inject - Pressure    2.5 psi    10 sec    BI: B1    BO: A1    forward    0.00    Separation - Voltage    15 kV  0.5 psi    60.00 min    BI: B1    BO: A1    1.0 min ramp, normal
  polarity, both    1.00    Relay On                             60.00    Separation - Voltage    1.0 kV&nbsp;   5 psi    5.00 min    BI: B1    BO: A1    5.0 min ramp, normal
  polarity, both    65.00    End
?. Eluted proteins were ionized in positive ion mode nanoelectrospray ionization (nESI) using a sheathless emitter.High-HighSpray voltage1400 - 1800Sweep gas0Ion transfer tube temp320Application modeIntact ProteinPressure modeLow PressureAdvanced Peak DeterminationTrueDefault charge state15S-lens RF30Source fragmentation15 eV
High-HighSpray voltage1400 - 1800Sweep gas0Ion transfer tube temp320Application modeIntact ProteinPressure modeLow PressureAdvanced Peak DeterminationTrueDefault charge state15S-lens RF30Source fragmentation15 eV
?. Precursor (intact protein) spectra were acquired at 120k FTRP.High-HighDetector typeOrbitrapResolving power120000m/z RP measured200 m/zScan range450-2000Mass rangeNormalAGC target2000000Normalized AGC target500%Max Injection Time50 msMicroscans1Data typeProfilePolarityPositiveUse wide quad isolationTrue
High-HighDetector typeOrbitrapResolving power120000m/z RP measured200 m/zScan range450-2000Mass rangeNormalAGC target2000000Normalized AGC target500%Max Injection Time50 msMicroscans1Data typeProfilePolarityPositiveUse wide quad isolationTrue
?. The mass spectrometer was operated using a TopN 3 sec data-dependent acquisition modePrecursor ions were filtered by intensity, charge state, and dynamic exclusion:Intensity minimum5000Intensity maximum1E20Included charge states4-60Include undetermined charge statesFalseDynamic exclusion after n times1Dynamic exclusion duration45 sMass tolerance0.5 m/zExclude isotopesTrue
Intensity minimum5000Intensity maximum1E20Included charge states4-60Include undetermined charge statesFalseDynamic exclusion after n times1Dynamic exclusion duration45 sMass tolerance0.5 m/zExclude isotopesTrue
?. Ions for fragmentation were isolated and fragmented via higher energy dissociation (HCD):High-HighDetector typeOrbitrapIsolation modeQuadrupoleResolving power60000m/z RP measured200 m/zScan range350-2000AGC target400000Normalized AGC target800%Max injection time600 msMicroscans1Isolation window3 m/zActivation typeHCDCollision energy32Collision energy modeFixedPolarityPositive
High-HighDetector typeOrbitrapIsolation modeQuadrupoleResolving power60000m/z RP measured200 m/zScan range350-2000AGC target400000Normalized AGC target800%Max injection time600 msMicroscans1Isolation window3 m/zActivation typeHCDCollision energy32Collision energy modeFixedPolarityPositive
?. Setup buffers in tray:LocationBuffer compositionBI:A1Background electrolyteBI:B1Background electrolyteBI:C10.1 M HClBI:D1WaterBO:A1Conductive liquidBO:B1Water
LocationBuffer compositionBI:A1Background electrolyteBI:B1Background electrolyteBI:C10.1 M HClBI:D1WaterBO:A1Conductive liquidBO:B1Water
?. Prior to first use, the CESI cartridge were conditioned. This process can be divided into two methods: 1) washing method, 2) electrical conditioning method. Each morning, a shortened version of the electrical conditioning method was performed (+15 kV for 30 min). For long term storage, capillaries were rinsed with water and kept at 4 °C.Time [min]EventValueDurationInletOutletSummaryRinse - Pressure100 psi5.00 minBI: C1BO: B1forwardRinse - Pressure100 psi10.00 minBI: A1BO: B1forwardRinse - Pressure100 psi5.00 minBI: D1BO: B1reverseRinse - Pressure100 psi30.0 minBI: D1BO: B1forwardTime [min]EventValueDurationInletOutletSummaryRinse - Pressure100 psi3.00 minBI: A1BO: A1reverseRinse - Pressure100 psi5.00 minBI: A1BO: A1forward0.00Separate - Voltage15.0 kV60.00 minBI: B1BO: A11.00 min ramp, normal polarity, both60.00Separate - Voltage1.0 kV5.00 minBI: B1BO: A15.00 min ramp, normal polarity, both65.00End
Time [min]EventValueDurationInletOutletSummaryRinse - Pressure100 psi5.00 minBI: C1BO: B1forwardRinse - Pressure100 psi10.00 minBI: A1BO: B1forwardRinse - Pressure100 psi5.00 minBI: D1BO: B1reverseRinse - Pressure100 psi30.0 minBI: D1BO: B1forward
Time [min]EventValueDurationInletOutletSummaryRinse - Pressure100 psi3.00 minBI: A1BO: A1reverseRinse - Pressure100 psi5.00 minBI: A1BO: A1forward0.00Separate - Voltage15.0 kV60.00 minBI: B1BO: A11.00 min ramp, normal polarity, both60.00Separate - Voltage1.0 kV5.00 minBI: B1BO: A15.00 min ramp, normal polarity, both65.00End