The dried-droplet method is the oldest and has remained the preferred sample preparation method in the MALDI community.
1. Prepare a fresh saturated solution of matrix material in the solvent system of choice: A small amount,10-20 mg, of matrix powder is thoroughly mixed with ? 1mL of solvent in a 1.5 mL Eppendorf tube, and then centrifuged to pellet the undissolved matrix.
2. Place 5-10 mL of the supernatant matrix solution in a small Eppendorf tube. (Note: Typical concentrations in saturated matrix-only solutions are in the 1-10 millimolar range.)
3. Add a smaller volume (1 to 2 mL) of protein solution (1-100 mM) to the matrix.
4. Mix the solution thoroughly for a few seconds in a vortex mixer.
5. Place a 0.5-2 uL droplet of the resulting mixture on the mass spectrometer’s sample stage.
6. Dry the droplet at room temperature. (Note: Blowing room-temperature air over the droplet speeds drying.)
7. When the liquid has completely evaporated, the sample may be loaded into the mass spectrometer. Typical analyte amounts on MALDI crystalline deposits are in the 0.1-10 picomole range.
Crystal washing: The analyte/matrix crystals may be washed to etch away the involatile components of the original solution that tend to accumulate on the surface layer of the crystals (segregation). The procedure most often recommended is to thoroughly dry the sample (dessicator or vacuum dry) followed by a brief immersion in cold water (10 to 30 seconds in 4? C water.) The excess water is removed immediately after by flicking the sample stage or by suction.
This method is surprisingly simple and provides good results for many different types of samples. Dried droplets are very stable and can be kept in vacuum or in a dark drawer for days before running a MALDI experiment.
The dried-droplet method tolerates the presence of salts and buffers very well, but this tolerance has its limits. Washing the sample as described above can help; however, if signal suppression is suspected, a different approach should be tried (see crushed-crystal).
The dried-droplet method is usually a good choice for samples containing more than one protein or peptide component. The thorough mixing of the matrix and analyte prior to crystallization usually assures the best possible reproducibility of results for mixtures.
A common problem in the dried droplet method is the aggregation of higher amounts of analyte/matrix crystals in a ring around the edge of the drop. Normally these crystals are inhomogeneous and irregularly distributed, which is the reason MALDI users often end up searching for “sweet spots” on their sample surfaces. As an example, it has been observed [ ] that peptides and proteins tend to associate with the big crystals of 2,5-dihydroxybenzoic acid that form at the periphery of air dried drops containing aqueous solvent, whereas the salts are predominantly found in the smaller crystals formed in the center of the sample spot at the end of crystallization. In a clever set of experiments, Liang Li and co-workers [ ] used confocal fluorescence to demonstrate that with the dried-droplet method, the analyte is not uniformly distributed among or within the matrix crystals. In fact, some crystals show no analyte at all. Most well-written MALDI software packages allow for automated sweet-spot searching during data acquisition, a procedure by which the sample surface is scanned with the laser beam until a portion yielding strong signals is located.
Another problem that is often observed during crystallization is what is known as segregation: as the solvent evaporates and the matrix crystallizes, the salts and some of the analyte are excluded from matrix crystals. This is particularly important in cases where cationization is the ionization mechanism, such as in the case of synthetic polymers and carbohydrates. Component segregation yields an inhomogeneous mixture of analyte throughout the sample, resulting in highly variable analyte ion production as the laser is moved across the sample surface.