Mass Spectrometry Laboratory

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Electrospray Ionization (ESI) Instructions

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.

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, sodium, Na, can be introduced even from glassware. Sulfate and phosphate impurities in protein samples can lead to an adduct with mass of 98 m/z. 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. The sample should be clear. Microscopic debris can clog the electrospray needle.

Getting an ESI Signal

Login to your account. Write the information needed in the Log Book. Open "apexControl Standard" software.

Load the sample or standard into the 100 μL or 250 μL 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. Load calibrated 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 μA) 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. Set the number of scans to average 10-50.

The "Tune" mode allows the operator to optimize signal.

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

Mass Calibration

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 - minimum 4 peaks. Click "Accept". To check this calibration, click "Automatic". Repeat this process until the entire mass range is calibrated.