Back to the Lab!

On Friday (April 4th), I finally returned to the lab at RPI after a month of break! Today I took a break from my project to get some experience with MALDI. MALDI is matrix-assisted laser desorption/ionization. This process uses mass/charge ratio of ions to see if a peptide was synthesized correctly. First, I put peptides in solution by adding 100 microliters of acetonitrile-water mixture to each of the six sample. I then added 10 microliters of CHCA and 10 microliters of peptide solution to smaller vials.

We then spotted 1 microliter spots onto a steel MALDI plate. Spots A1 and B1 were CHCA and standards, spots A2 and B2 were sample one, spots A3 and B3 were sample 2, etc.

In the MALDI machine, a laser is used to strike a small area of the sample spot. The sample then ionizes into gases. In the system we are using, differently charged plates serve to shoot the ions down the tube, where there are other differently charged plates at the end. Depending on the size and charge of the ions, the ions go different distances into the field of plates at the end of the tube before they are reflected back towards the reflection detector. Because larger ions go deeper into the end plate area, they take longer to get back to the detector, and the detector can give information about the mass/charge ratio of the ions. This process can be seen in the diagram below.

Image by JP Trasatti, 4/4/2014

To shoot the laser, you first choose a part of the sample to shoot because the laser area is much smaller than the area of the spot. The computer program magnifies the sample plate and shows the peptide as white spots within the sample spot. You take multiple samples of each spot to minimize the baseline and increase the signal from the laser.

After finishing the MALDI process, I helped JP check on the brain cell samples and check the CO2 content of the incubator. To check the CO2 level, we used a device that measures the CO2 with a KOH solution. This device attaches to a connector on the incubator with a tube. There is a pump on the tube that you squeeze 18 times to pump a sample of the incubator air into the device. The device is then rotated so the gas combines with the KOH solution. The CO2 dissolves in the KOH solution, increasing the volume of the liquid in the device to a measurable level. We want the concentration to be at 5.0 in the incubator, and we found it to be 5.2, so we recalibrated the incubator to decrease the amount of CO2.