Preparation of Homogeneous MALDI Samples for Quantitative Applications

The overall goal of this sample preparation method from MALDI MS is to reduce the spatial heterogeneity in ion signals during MALDI MS.A well controlled drying environment was built to optimize the conventional dried-droplet method, by manipulating the hydrodynamic flows inside the sample droplet during the drying process. The main advantage of this technique is that the spatial heterogeneity of the ion signals for the sample prepared by the optimized dried-droplet method can be effectively reduced. Though this method can provide homogenous samples for the THAP Metric System.

It can also be applied to other metric systems, such as alpha-Cyano-4-hydroxycinnamic acid. Generally, individuals new to this method were struggled because controlling the drying condition is not easy. To begin this procedure, open the door of a drying chamber and immediately place a previously cleaned sample plate on the copper-based block.

Then, close the door. Manually adjust the gas-flow meter to set the nitrogen flow rate to 10 standard cubic feet per hour. Following this, fine-tune the gas-flow meter to ensure the relative humidity is always below 25%in the drying chamber, as measured by a hygrometer.

Using K Type Thermocouples to monitor the temperature, adjust the water circulator temperature manually, until the sample plate reaches 5 degrees Celsius for the experiment. Ensure the required temperatures and the relative humidity are reached before sample deposition. Mix 0.25 microliters of 0.1 Molar THAP solution and 0.25 microliters of the desired analyte in a microcentrifuge tube.

Vortex the sample for three seconds. Then, centrifuge the sample for two seconds to collect the solution at the bottom of the microcentrifuge tube. Following centrifugation, open the door of the drying chamber and carefully deposit 0.1 microliters of the solution on the sample plate with a pipette.

Close the door immediately and wait for the sample droplet to dry out. When keeping the sample plate temperature lower than its surroundings, the average velocity of recirculation flows within the droplet is significantly increased to redistribute molecules. Sample deposition is the most critical step that determines data quality and reproducibility.

The premixed sample solution needs to be deposit immediately on the sample plate to ensure the sample is crystallized under controlled condition. After drying, open the door. of the drying chamber.

Then, set the water circulator temperature to 25 degrees Celsius. Once the sample plate temperature returns to 25 degrees Celsius, remove the sample plate from the drying chamber. Examine the sample morphology under a five times magnified Stereo microscope, and take a snapshot brightfield image.

At this point, insert the sample plate into a MALDI Mass Spectrometer. Then, perform imaging mass spectrometry analysis. Select a characteristic mass peak from the mass list shown in the result window and click 2D to plot a two-dimensional ion image.

Next, click the adjustment buttons in the pop-up window to determine the upper and lower limits of the signal intensity, to increase the image resolution, and click save a picture. Observe and compare the ion image with the previously acquired brightfield image to obtain the essential sample region. Now, click the null spots and the cracked regions in the ion image shown in the result window to fine-adjust the region to be analyzed.

Click the find edge button to find the outer-most layer of the ion image. Following this, click deduct to save the ion abundance information of the outmost layer in a database, and remove this layer from the ion image simultaneously. After repeating the previous steps until the center of the ion image is defined, click and select all the checkboxes in the output data list and click export to export the data.

Finally, open the exported data using spreadsheet software to calculate the average ion abundance of every layer, to obtain the spatial distribution information of ions. In the brightfield and MALDI images of maltotriose, the ion signal of the sodiated form mainly populates at the periphery of the sample area, when it is prepared with a sample plate temperature of 25 degrees Celsius. By decreasing the temperature to five degrees Celsius, the signal populates homogeneously over the entire sample area, suggesting that preparing samples under a lower sample plate temperature can significantly redistribute the molecules and reduce heterogeneity.

The protonated bradykinin fragment ion image shows a similar trend as the sodiated maltotriose. Statistical analyses show that the heterogeneity of ion signals obtained under a sample plate temperature of 5 degrees Celsius reduces by 60 to 80 percent, with respect to that under 25 degrees Celsius. In the case of sodiated maltotriose, with a sample plate temperature of 25 degrees Celsius, the signal intensities at the centers are much lower than those with the temperature of 5 degrees Celsius.

The protonated bradykinin fragment shows less variation when decreasing the sample plate temperature from 25 to 5 degrees Celsius. While attempting this drying method, the temperatures and relative humidity in the drying chamber need to be carefully controlled and monitored. Once mastered, a sample prepared by this technique can be done in 30 minutes if it is performed properly.

After it's development, this technique paved the way for researchers in the field of MALDI MS to explore quantitative analysis for dried-droplet samples. After watching this video, you should have a good understanding of how to prepare homogeneous dried-droplet samples for MALDI MS analysis.