Bioanalytical Research

Bioanalytical Research Group

MALDI Sample Preparation - Dried Droplet Method

This is the original sample preparation method for MALDI MS. The sample (peptide or protein solution, in this case) is mixed with the matrix solution, deposited on the MS probe, and dried. This protocol works well for sinapinic acid and HCCA matrices.

1. Make a saturated solution (~ 10 g/L) of matrix

by adding one volume acetonitrile and two volumes of 0.1% TFA to an aliquot of matrix (dry powder). Vortex for at least 30 seconds. The solution should appear slightly turbid. Centrifuge to precipitate insoluble matrix material. Use only the supernatant in subsequent steps. The matrix solution can be used for several days. Alternatively, ready-made matrices can be purchased from some MALDI MS manufacturers. 

2. Dilute protein samples

to a final concentration of 5-50 µM in 0.1% TFA, if possible. Dilute peptide samples to a final concentration of 0.5-10 µM in 0.1% TFA, if possible.

3. Mix 1 µL of sample solution into 9 µL matrix solution

in a microcentrifuge tube and vortex. The mixing ratio of analyte solution to matrix solution can be varied from 1:1 to 1:9 (v/v) depending on the amount of analyte available. Use a 1:1 ratio for less abundant samples.

4. Deposit

0.5-1 µL of the analyte/matrix solution onto the mass spectrometer probe.

5. Dry the droplet

in a stream of air or nitrogen at ambient temperature (do NOT use heat!)..

6. Cleaning

If interfering substances are expected to be present the sample deposit can be cleaned by gently "etching" the surface of the crystals with cold water or 0.1% TFA (64). Dip the probe tip into cold water or add a drop of cold water on top of the sample deposit, followed by wiping or shaking off the water.

Insert the probe into the MALDI mass spectrometer.

Comments For difficult samples (e.g. hydrophobic peptides and proteins), 5-33% formic acid can be used instead of 0.1% TFA and isopropanol can be used instead of acetonitrile. Sinapinic acid and HCCA are good matrices combined with high acid concentrations.