1. Sample spotting techniques Dried droplet Crushed crystal Thin layer Sandwich

2. Dried Droplet Most common method used with all matrices. Premix sample and matrix solution in small vial (500 µL Eppendorf tube), spot 0.5 - 1 µL onto sample plate and air dry. For small sample amounts, deposit 0.5 µL on plate, then add 0.5 µL matrix on top; mix and allow to dry.

3. Crushed Crystal Method Prepare a saturated solution of sinapinic acid (or CHCA) in 30% AcN. Deposit 1 µL of this solution on the sample plate and allow to dry. Crush the crystals formed with a spatula or a foil- wrapped pencil eraser. Add 0.5 - 1 µL of sample in saturated matrix on top of the crystal layer and allow to dry.

4. Thin Layer Use 10 mg/mL CHCA in acetone. Apply 0.5 µL to plate and dry. Place 0.5 µL sample in 0.1% TFA on top of the matrix layer and allow to dry. If sample is not acidified, 0.5 µL 0.1% TFA can be added to the plate, then the sample.

5. Sandwich Method Deposit 0.5 µL matrix(in acetone or methanol) on plate and let dry. Add 0.5 µL sample in 0.1% TFA, then 0.5 µL matrix (in 50% Acn with 0.1% TFA) and dry in air.

6. Ideal Sample Concentrations Peptides and proteins: 0.1 to 10 µM Oligonucleotides: 10 to 100 µM Polymers: 100 µM