Electrospray Ionization (ESI) Instructions

  1. General Information, Guidelines, and Sample Preparation
  2. Getting an ESI Signal
  3. Mass Calibration

  1. General Information, Guidelines, and Sample Preparation
  2. Solvent selection. The solvent should ensure that the analyte is susceptible to charging. Acid in concentration of 0.1% is often added. Solution must be conductive.

    For proteins: 1:1 water and methanol + 0.1% formic or acetic acid.

    ESI compatible solvents: water, acetonitrile, methanol, dichloromethane, DMSO, isopropanol, butanol, THF, acetone, DMF.

    ESI compatible buffers: acetic acid, formic acid, ammonium acetate, ammonium hydroxide.

    These do not work well for ESI: hydrocarbons, aromatics, carbon tetrachloride, toluene.

    Solvent purity. Deionized water with conductivity > 18 Ω/cm3 should be used.

    Impurities in organic solvents can result in unwanted peaks. For example, the plasticizer bis(ethylhexyl)phthalate appears in mass spectra as [M+H]+ at m/z 391, or [M+Na]+ at m/z 413.

    Sodium, Na, can be introduced even from glassware.

    Sulfate and phosphate impurities in protein samples can lead to an adduct with mass of 98.

    Common surfactants, such as sodium dodecyl sulfate (SDS) and Triton X in protein samples can completely mask the protein signal.

    Sample concentration. The sample concentration should be in the range of 0.5-5 μM.

    Filtration. The sample should be clear. Microscopic debris can clog the electrospray needle.

  3. Getting an ESI Signal
  4. Login to your account. Write the information needed in the Log Book.

    Open "apexControl Standard" software.

    Load the sample or standard into the 100 mL or 250 mL syringe.

    Set the pump flow to 120-300 μL/hr, connect the syringe to the injection port and turn on the flow. Fast forward the sample through the tubing to remove any air in the system.

    Make sure that the BEAM VALVE is open.

    Load previously created ESI method or open a previously acquired file. This will load a set of instrumental parameters.

    Make sure that there is capillary current (15-20 mA) present - in the "Readback" tab. If there is no current, check if the "Nebulizer Gas Flow", "Drying Gas Flow", "ESI High Voltage", and the "Drying Gas Heater" are turned on, and the "Dry Temp" set to ñC.

    Set the number of scans to average 10-50.

    The "Tune" mode allows the operator to optimize signal, resolution, and MS/MS fragmentation in real time.

    In order to acquire a data file, edit the "Prefix" field and the "Subdirectory" field. Click "Acquisition/Run Method" to acquire the spectrum.

  5. Mass Calibration
  6. Once a spectrum has been acquired, the FT-ICR must be calibrated.

    Open the correct mass list.

    Select the calibration mode - Cal2 or Cal3.

    Select the peak to be calibrated in the "Current Mass" column of the corresponding reference mass. For Cal2, a minimum of 3 peaks are required. For Cal3 - min 4 peaks. Click "Accept". To check this calibration, click "Automatic".

    Repeat this process until the entire mass range is calibrated.