An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

A simple sample preparation procedure was developed to improve the conventional method in matrix-assisted laser desorption ionization mass spectrometry by reforming the simple morphology during the drying process. The advantage of this technique is that the signal intensity of carbohydrate samples prepared by this optimized method can be effectively enhanced. Generally, individuals new to this method will struggle because the methanol depressurization step is very crucial and it needs to be done in one go.

Any hesitation will decrease the data quality. To begin, while wearing nitrile gloves, use 100 milliliters of detergent solution to hand wash the sample plate. Use distilled-deionized, or DD, water to hand wash the plate.

Then, with 30 milliliters of methanol, rinse its surface. Insert the sample plate into a 600 milliliter beaker and fill it with DD water until the plate is fully immersed. Then place the beaker into an ultrasonic bath and sonicate the plate for 15 minutes.

Remove the sample plate from the beaker and use pressurized nitrogen to blow off the water drops. Then deposit 0.2 microliters of methanol on the sample plate to check whether it spreads to other spots. If it does, repeat the wash, sonication and nitrogen treatment as just demonstrated.

After preparing the drying chamber according to the text protocol, premix 0.25 microliters of 2, 5-Dihydroxybenzoic acid solution and 0.25 microliters of Sialyl-Lewis A, or maltoheptaose solution in the micro centrifuge tube. Then vortex the tube for three seconds. Spin down the mixed solution in the mini centrifuge at 2000 times G for two seconds.

Then immediately pipette 0.1 microliter of the pre-mixed solution on to the sample plate. When the sample has dried, while working quickly to avoid evaporation, pipette 0.2 microliters of methanol directly on to the sample spot. The sample will redissolve and then immediately dry out.

The methanol deposition step is the most difficult step that determines data quality and reproducibility. To ensure the best performance, we recommend that the user finish the deposition in three to five seconds to avoid significant evaporation loss of methanol. Wear nitrile gloves and carefully remove the sample plate from the drying chamber.

Then, under a microscope, examine the sample. If the crystal morphologies are not as expected, repeat mixing the sample and spotting onto the sample plate. To ensure the sample crystals have the optimal morphology after recrystallization, always use a microscope to exam them.

It is difficult to determine the quality of crystal morphology correctly with the naked eye. To analyze a one microliter sample, premix 2.5 microliters of 2, 5-Dihydroxybenzoic acid solution and 2.5 microliters of Sialyl-Lewis A or maltoheptaose solution in a micro centrifuge tube. Vortex the pre-mixed solution for five seconds.

Spin down the mixed solution at 2000 times G for two seconds, then immediately pipette one microliter of the pre-mixed solution on to the sample plate. After the sample dries out, pipette 1.5 microliters of methanol directly on to the dried sample spot. The sample will redissolve and immediately dry out.

Carefully remove the sample from the drying chamber. Examine the sample under a microscope. If the crystal morphologies are not as expected, repeat the solution preparation as just demonstrated.

To carry out mass spectrometry, open the mass spectrometer control software and insert the sample plate into the machine. Select the pre-optimized data acquisition method in the software, then using the imaging software, register the whole sample region for imaging mass spectrometry. Start data acquisition in batch mode of the control software.

When data acquisition is complete, use the imaging software to plot the ion images and analyze the data according to the text protocol. Representative SEM images of Sialyl-Lewis A mixed with 2, 5-Dihydroxybenzoic acid prepared using dried droplet and recrystallization methods are shown here. A typical 2, 5-Dihydroxybenzoic acid morphology is large, needle-shaped crystals at the rim and fine crystalline structures in the center of sample spots.

After recrystallization by methanol, the sample has a larger area covered evenly with fine, flake-like crystals. This figure depicts imaging mass spectrometry results of Sialyl-Lewis A and maltoheptaose with and without methanol recrystallization. After recrystallization, the distribution of Sialyl-Lewis A and maltohepaose signals match well with the bright-field images of sample spots.

The signal intensities of the recrystallized carbohydrate samples are also enhanced compared to conventional dry droplet samples. This graph compares the signal intensity of sodiated or positive ion mode and deprotonated or negative ion mode carbohydrates of recrystallized samples with respect to that of dried droplet samples. On average, recrystallization of Sialyl-Lewis A and maltoheptaose samples increased sodiated signals by factors of 3.9 and 3.3.

Deprotonated Sialyl-Lewis A ion signals were enhanced by a factor of 4.7 after recrystallization. Once mastered, samples can be prepared in 10 minutes using this technique if it is performed properly. While attempting this procedure, it's important that the methanol droplet be immediately and precisely deposited on the sample spot.

After watching this video, you should have a good understanding of how to effectively control crystal morphology of multi samples to obtain the best ion signals for routine analysis.