The crushed-crystal method  was specifically developed to allow for the growth of analyte doped matrix crystals in the presence of high concentrations of involatile solvents (i.e. glycerol, 6M urea, DMSO, etc.) without any purification.
1. A fresh saturated solution of matrix material in the solvent system of choice is prepared in the same fashion as in step 1 of the dried-droplet method. The supernatant liquid is transferred to a separate container before use to eliminate the potential presence of undisolved matrix crystals.
2. An aliquot (5 to 10 mL) of the saturated matrix solution is mixed with the protein containing solution (1 to 2 mL) to produce a final protein concentration of 0.1 – 10 mM. This analyte/matrix solution is equivalent to the one that would be made in the simpler dried-droplet experiment. Note: particular attention must be paid to eliminate the presence of particulate matter in this solution. Centrifuge, and use the supernatant, if necessary.
3. A 1 mL drop of the matrix-only solution is placed on the sample stage and dried in air. The deposit formed looks identical to what is typically obtained from a dried-droplet deposit.
4. A clean glass slide (or the flat end of a glass rod) is placed on the deposit and pressed down on to the surface with an elastic rod such as a pencil eraser. The glass surface is turned laterally several times to smear the deposit into the surface.
5. The crushed matrix is then brushed with a tissue to remove any excess particles (no need to be particularly gentle)
6. A 1 mL droplet of the analyte/matrix solution is then applied to the to the spot bearing the smeared matrix material.
7. Within a few seconds an opaque film forms over the substrate surface covering the metal.
8. After about 1 minute the sample is immersed in room temperature water to remove involatile solvents and other contaminants. Note that it is not necessary to let the droplet dry before washing: the film does not wash off easily.
9. The film is blotted with a tissue to remove excess water and allowed to dry before loading into the mass spectrometer.
The dried-droplet method is widely used because it is simple and effective. Good signals are obtained from initial solutions that contain relatively high concentrations of contaminants (salts and buffers). Many real analytical samples contain those materials and the capacity to tolerate these impurities has an enormous practical importance. However, there are limits to the contamination tolerance of the dried-droplet method. Particularly, the presence of significant concentrations of involatile solvents reduces, or totally eliminates, the ion signals. Examples of the most common of these solvents are dimethylsulfoxide, glycerol and urea. Removal of the involatile solvents may not be possible if they are needed to disolve or stabilize the analyte.
The dried-droplet method forms crystals randomly throughout the droplet as the solvent evaporates. The surface of the droplet is the preferred site for initial crystal formation. The crystals form at the liquid/air interface and are then carried into the bulk of the solution by convection. The final sample deposit is littered with those crystals, and if no involatile solvent is present they become adhered to the substrate. If involatile solvents are present, the crystals might either not form or remain coated with the solvent, preventing them from attaching to the substrate. Even if crystals are formed and the deposit is introduced into the mass spectrometer, a coating of involatile solvent usually suppresses the ion signals. Attempts to wash the crystals usually results in their loss, because they are not securely bonded to the substrate.
The crushed-crystal method is operationally similar to the dried-droplet method, but the results are very different, particularly in the presence of involatile solvents. In this method rapid crystallization directly on the metal surface is seeded by the nucleation sites provided by the smeared matrix bed that is crushed on the metal plate prior to sample application. Crystal nucleation shifts from the air/liquid interface to the surface of the substrate and microcrystals form inside the solution where the concentrations change slower. The polycrystalline film adheres to the surface so the crystallization can be halted any time by washing off the droplet before its volume decreases significantly.
The films produced are also more uniform than dried droplet deposits, with respect to ion production and spot-to-spot reproducibility.
The disadvantages of the crushed-crystal method are the increase in sample preparation time caused by the additional steps. It does not lend itself to automation for high throughput applications. It requires strict particulate control during solution preparation to eliminate the presence of undisolved matrix crystals that can shift the nucleation from the metal surface to the bulk of the droplet.
The crushed-crystal metod has been succesfully applied to the analysis of proteins as well as oligonucleotides . Variations on this method have also been introduced in the literature .