Welcome to the CHI Lab tutorial on the ultra thin layer method, which is particularly useful for the accurate mass determination of proteins. Because of the sensitive nature of mass spectrometry, we only use HPLC grade reagents as well as powder free latex or nitrile gloves To clean the Plate. Rinse alternately with methanol water, and then finally methanol again.
In between each rinse, gently wipe away the excess solvent using lens tissue. You may repeat these wash steps as many times as necessary to produce a clean plate. And once cleaned and dry, the plate should be used Immediately.
Apply and spread 20 to 30 microliters of thin layer substrate solution across the clean MALDI plate. Here we use a P two 50 pipette tip to evenly spread the solution while avoiding scratching The plate. As the thin layer Dries, remove any remaining water droplets if present by gently blotting with a kim wipe.
Then gently wipe the plate in one smooth motion with a Kim wipe to properly form the thin layer. Finally, clean the edges of the plate to avoid contaminating the Mass spectrometer. It is crucial to Test the thin layer before applying your sample.
If the test spot takes too long to dry where the crystals are not homogenous, the problem may lie Within the thin layer itself or the matrix solution, clean the plate, reapply the thin layer, and retest. When spotting onto The thin layer, you do not want your spots to evaporate to dryness. Rather, once crystallization has slowed and stopped, remove the excess solvent.
Using a vacuum aspirator, The Presence of contaminants such as salts and detergents, as well as high analyte concentration may interfere with crystallization. Further sample dilution may improve crystallization Spot the Ants in close proximity to the sample. This will allow for pseudo internal calibration in which the plate is moved from the sample spot to the calibrate spot.
During sample acquisition, adding a few laser shots of the CAS to the spectrum, this approach guarantees a higher mass accuracy calibration than external calibration alone. It is very important to wash away any contaminants such as salt or detergent that may interfere with analyte.Ionization. To do so, drop ice cold, 0.1%Tri Fluor acetic acid in HPLC, grade water directly onto the spots.
Remove the wash solution using a vacuum Aspirator As shown here. This method produces excellent spectra for both membrane and soluble proteins.
