The discovery of this method allowed the application of laser desorption to proteins [2].Drying a droplet of a protein/matrix solution remains the favorite method of most MALDI practitioners.The recipe for producing a dried droplet sample has several simple steps.First,make a saturated solution of the matrix material (see Table 1 for solvents)and mix in sufficient protein for a final concentration of 1-10 mM.This solution must be thoroughly mixed to ensure reproducible results.A convenient method is to place some saturated matrix solution (5-10 microliters)in an Eppendorf tube and then add a smaller volume (1-2 microliters)of a protein solution.Agitating the tube with a vortex mixer for a few seconds mixes the solution sufficiently.Second,place a droplet (0.5-2 microliters)of the resulting mixture on the mass spectrometer's sample stage.Dry the droplet at room temperature; blowing room-temperature air over the droplet speeds drying.When the liquid has completely evaporated,the sample may be loaded into the mass spectrometer.There is no rush to load the sample.Dried droplets are quite stable: they can be kept in a drawer or in vacuum for days.The deposit may also be washed to etch the surface layer of the deposit's crystals; the surface layer is the most heavily contaminated with involatile components of the original solution.Be careful when etching the crystals as it is easy to wash them off the surface.We recommend thoroughly drying the sample (vacuum-dried if possible)followed by a brief immersion in cold water (10 - 30 s in 4?C water).The excess water should be removed rapidly (e.g.,by flicking the sample stage,or by suction).

Some authors suggest placing a drop of saturated matrix solution on the sample stage and then adding a similar volume of protein solution to form the final droplet.This method results in acceptable spectra for samples containing a single analyte.If the protein sample contains more than one protein or peptide component,we recommend thorough mixing of the two solutions in a tube before making the droplet.Prior,thorough mixing increases the reproducibility of the mass spectra obtained.

The simplicity of the dried droplet recipe surprises most people.This method gives good results for many types of protein samples.The recipe has some serious pitfalls that the unwary may encounter.

The protein must be truly dissolved in the solvent.Making a slurry of a peptide powder and solvent is not sufficient.

Be careful of inadvertent changes in solvent composition.Such changes can easily occur when two solutions are mixed,resulting in the precipitation of either the protein or matrix (or both).A similar problem can result from the selective evaporat ion of organic solvents from aqueous solutions.The latter is a particular problem when small volumes of solution are stored in relatively large containers (e.g.,10 microliters of solvent in a 1.5 ml Eppendorf tube).

Do not heat the droplet to speed drying.Changing the solution's temperature alters matrix crystal formation and protein incorporation,usually in a bad way.

Use fresh matrix solutions whenever possible.Matrix solutions gradually decompose under normal laboratory conditions.Mix up small volumes of solution as needed.

Do not use higher protein concentrations than recommended: the final protein concentration should be less than 10 micromolar.It is tempting to add more protein,in the hopes of increasing signal intensity.This approach seldom works.Reducing the protein concentration (not increasing it)frequently increases the signal.

Do not use involatile solvents.The involatile solvents commonly used in protein chemistry are glycerol,polyethylene glycol,b-mercaptoethanol,Triton-X,dimethylsulfoxide (DMSO)and dimethylformamide (DMF).These solvents interfere with matrix cry stallization and coat any crystals that do form with a difficult-to-remove solvent layer.If you cannot avoid using these solvents (or if a droplet does not dry properly),try another method (see below).

Keep the concentration of non-protein materials to a minimum.The dried droplet method tolerates the presence of salts and buffers very well,but this tolerance has limits.Washing the crystals may help,although care must be taken not to wash the c rystals off the substrate.If you suspect that contaminants are suppressing your signal,try another method (see below).

The crystal growing solution's pH must be less than 4.The organic acid matrices described above become ions at pH > 4,completely changing their crystallization properties.Using aqueous 0.1% trifluoroacetic acid rather than pure water will usually solve any pH problems.

Do not leave crystals of undissolved matrix in the final protein/matrix solution.Intact matrix crystals act as crystal seeds,leading to very inhomogeneous samples.Spinning the matrix solution in a table-top centrifuge removes the large crystals t hat cause this problem.