Bioanalytical Research

Bioanalytical Research Group

This procedure allows the matrix and the sample to be applied onto the MS probe in independent steps. This simplifies sample preparation and also improves sensitivity and resolution attainable in peptide analysis (refer to publications cited below).

1. Standard method:

Make a saturated solution (~ 40 g/L) of matrix in acetone in a 1.5 mL microcentrifuge tube. Vortex briefly. Centrifuge to precipitate insoluble matrix material. 

Note: The matrix has to be soluble in acetone or some other fast evaporating solvent. Sinapinic acid, ferulic acid, HCCA, and THAP (see Table of Matrices) are routinely used.

2. Method for low-level and "dirty" peptide samples:

Make a 10 g/L solution of nitrocellulose in acetone/isopropanol (1:1). Mix nitrocellulose solution in a 1:4 ratio (v/v) with matrix/acetone solution (see step 1a). The nitrocellulose makes the fast evaporation matrix film more robust towards contaminants and allows thorough washing of the sample prior to analysis.

3. Prepare the sample

as for the dried droplet method. Further dilution is possible to obtain a 0.01_0.1 µM polypeptide solution which can help to reduce the concentration of contaminating species. Make sure that the sample solution is acidic (pH < 4) and contains less than 30% organic solvent so that the matrix film is not completely redissolved.

4. Deposit

0.5-1 µL sample solution on top of the thin film and allow it to dry at ambient temperature. If the sample contains more than 30% organic solvent, first deposit 0.5 µL of 5% formic acid on the film followed by injection of 0.5µL of the sample solution into this droplet.

5. Rinse the sample

by depositing 10-20 µL water or 1-5% formic acid on the probe followed by immediate wiping or shaking of the probe. This rinsing procedure can be repeated several times.

6. Insert the probe into the MALDI mass spectrometer.

References

• • "Matrix surfaces made by fast evaporation yield improved resolution and very high sensitivity in MALDI TOF", Vorm, O.; Roepstorff, P.; Mann, M. (1994) Analytical Chemistry, 66, 3281-3287.
• • "Improved mass accuracy in matrix assisted laser desorption/ionization time-of-flight mass spectrometry of peptides", Vorm, O.; Mann, M. (1994) J. Am. Soc. Mass Spec. 5 955-958.
• • "Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels", Shevchenko A., Wilm, M., Vorm, O. and Mann, M.(1996) Anal. Chem. 68, 850-858.
• • "Delayed extraction improves specificity in database searches by MALDI peptide maps", Jensen, O.N., Podtelejnikov, A., and Mann, M. (1996) Rapid Commun. Mass Spectrom., 10 1371 - 1378.