The overlayer method was developed on the basis of the crushed-crystal method and the fast-evaporation method. It involves the use of fast solvent evaporation to form the first layer of small crystals, followed by deposition of a mixture of matrix and analyte solution on top of the crystal layer (as in the sample matrix deposition step of the crushed-crystal method). The origin of this method, and its multiple names, can be traced back to the efforts of several research groups [References 8-10, 16].
1. First -layer solution (matrix only): Prepare a concentrated (5-50 mg/mL) matrix-only solution in a fast evaporating solvent such as acetone, methanol, or a combination of both.
2. Second-layer solution (analyte/matrix): Prepare the second-layer solution following the three steps below.
- 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 1 mL of solvent in a 1.5 mL Eppendorf tube, and then centrifuged to pellet the undissolved matrix. Place 5-10 uL of the supernatant matrix solution in a small Eppendorf tube.
- Add a smaller volume (1 to 2 uL) of protein solution (1-100 uM) to the matrix.
- Mix the solution thoroughly for a few seconds in a vortex mixer. This is the second-layer soluiton.
- Note: the solvent system for the second-layer must be chosen such that it does not completely dissolve the crystals when a drop of the solution is added to the dried first-layer deposit.
3. Apply a 0.5 uL drop of the first-layer solution to the sample plate and let it dry to form a microcrystalline layer.
4. Apply a 0.5-1 uL drop of the second-layer solution on top of the crystal bed and allow to air dry. Note: If the first crystal layer is completely dissolved, stop and retry using a smaller volume of second layer solution or a different solvent system.
Crystal Washing: Washing the crystals prior to introduction into the TOF spectrometer is often recommended. A large droplet of 5-10 uL of or dilute aqueous organic acid is applied on top of the sample spot. The liquid is left on the sample for 2-10 seconds and is then shaken off or blown off with pressurized air. The procedure can be repeated once or twice. The washing liquid must be free of alkali metals and should be neutral or acidic (i.e. 0.1% TFA).
The difference between the fast evaporation and the overlayer method is in the second-layer solution. The addition of matrix to the second step is believed to provide improved results, particularly for proteins and mixtures of peptides and proteins.
The overlayer method has several convenient features that make it a very popular approach
- It naturally inherits all the advantages detailed in the fast evaporation method, and it avoids some of its limitations.
- It provides enhanced sensitivity and excellent spot-to-spot reproducibility for proteins beyond what is possible with the fast-evaporation method. This enhancement is likely due to improved matrix isolation of the analyte molecules on the crystal surfaces in the presence of the surplus of matrix molecules.
- With the careful optimization of the second-layer analyte/matrix solution, the overlayer method is found to be very effective for the analysis of complicated mixtures containing both peptides and proteins. The ability to manipulate the second layer conditions adds flexibility to the sample preparation