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The sample preparation protocol for µFocusMALDI plate differs from conventional dried-droplet preparations. First, because spot sizes are much smallerthan conventional dried-droplet preparations, the matrix concentration should be reducedto a range between 1:5 and 1:50. Unlike conventional preparations, the µFocusMALDI plate preparation produces a concentration effect. As solvent is evaporated, the concentration of analyte, as well as the any contaminant present in the initial droplet, increases dramatically. Therefore, we recommend following the guidelines and protocols given below.

• The biochemical processing of analytesprior to MALDI preparation is crucial. Avoid detergents, contaminants or polymer releasing plastics.
  For in-gel digested samples, the protocol developed by Shevchenkohas been proved to be compatible with µFocusMALDI plates. (Shevchenkoet al., Anal. Chem. 68, 1996, 850-858).
• Using Incompatible plastics for µFocusMALDI plates may result in polymer release into the sample, leading to in improper or delayed crystallization of the matrix. On the other hand, some "high-grade" polypropylene tubes/ tips/ MTPsalso release polymers. Avoid using siliconizedtubes. We strongly recommend to use brands which are proven to be compatible with µFocusMALDI plate (please see the list of compatible chemicals and plastics).
• Solvent and matrix purity are of critical importance.
• Note : The analytes, along with any impurities present in the droplets, become concentrated during solvent evaporation. Avoid using detergents, as they temporarily eliminate the hydrophobic effect of the coatingand prevent droplets from evaporating. If solvent impurities cause problems, volume reduction or dilution in pure solvents is useful.
• In some cases, on-target washing and recrystallizationhelps eliminating salts and contaminants. On-target washing is also compatible with C18 ZipTipsample purification. (M. Schurenberget al. Anal. Chem. 72, 2000, 3436-3442).

α-cyano-4-hydroxycinnamic acid (HCCA) is water-insoluble and a widely used MALDI matrix for peptide analysis. The following protocol was specifically designed to maximize the use of HCCA on µFocusMALDI plate

Mix matrix and analytesolution in 1:1~5:1 ratio.		Loading 0.5 µl sample		Focusing Allow the sample to dry

Fig.1. The image above shows the focusing process of a sample droplet (1 µl of 0.3 mg/ml HCCA in EtOH/Acetone (2:1) applied to a µFocus MALDI plate.

• Matrix solution preparation:
  -Prepare 20 mg/ml HCCA dissolved in a mixture of acetonitrile(ACN)/0.1% trifluoroacetic acid (TFA) ; (1:1)
  -Incubate in a sonic bath for 5 min and centrifuge for 3~5 min at 8000~10000 rpm
  -Dilute saturated matrix solution (supernatant) 2~10 times with a solution of EtOH: Acetone (2:1)
• If the sample droplet contains more water, matrix crystallization starts earlier. If crystals are formed outside the anchor area, we recommend sample recrystallization.
• If you are working with a buffer-containing sample (e.g. in-gel digested samples with buffers) you may get improper crystallization. On-target washing and recrystallizationis recommended in this case.

Protein applications require higher organic solvent concentrations in sample droplets because hydrophobic domains of proteins tend to attach to the hydrophobic coating of µFocusMALDI plates.
This protocol was developed for small proteins (<20 kDa). Modifications may be required for larger proteins.

Mix matrix and analytesolution in 1:1~2:1 ratio.		Loading 0.5 µl sample		Focusing Allow the sample to dry

• Matrix solution preparation:
  -Prepare 2 mg/ml sinapinicacid (SA) dissolved in ACN/0.1% TFA (9:1).
  -Incubate in a sonic bath for 5 min and centrifuge for 3~5 min at 8000~10000 rpm

This process redissolvesmatrix crystals and concentrates the sample preparation onto the µFocusMALDI plate, independent of the initial droplet composition.

Dried sample with 'improper crystallization'		Apply 0.5~1 µl of EtOH/Acetone/ 0.1% TFA (6:3:1)		Allow the sample to dry

Principle : An aqueousdroplet containing analyteand contaminants (salt, buffer etc) is deposited onto the µFocusMALDI plate pre-covered with a thin layer of HCCA. The analytemolecules attach to the HCCA thin layer whereas the water soluble contaminants stay in the liquid phase and are removed together with the residual droplet after a few minutes. (Gobomet al., Anal. Chem. 73, 2001, 434-438).
Using this method, no further purification (e.g. ZipTip) is required even for crude samples. The thin layer affinity preparation is the method of choice for in-gel digested samples if no organic solvents are used when trypticfragments are eluted from the gel slices.

Apply 2 µl of matrix	immediately remove	Apply 0.5 µl aqueous analytesolution Incubation for about 1 min (the sample must not dry on the thin layer)		Add 2~4 µl of cold 1% TFA

Remove the whole droplet immediately

• Matrix solution: Saturated HCCA in acetone/0.1% TFA, (97:3)
• Analytesolution: 0.1~1% TFA (to maintain acidic conditions)
• Because the µFocusMALDI plate always retains 10~100 nlof ligand, the volume removed will be slightly lower than added. The addition of 1% TFA before removing the analytedroplet helps to dilute contaminants.

The following brands have been tested and proven to be compatible with µFocusMALDI plate.
Pipette tips: Eppendorfstandard tips
Vials/ tubes: Eppendorfsafe-lock
Bottles: NalgeneFEP (Teflon) bottles
MTPs: Greiner PP, natural, MTP

Solvents : HPLC grade or betterTroubleshooting