Laboratory of Mass Spectrometry and Gaseous Ion Chemistry
Brian T. Chait
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MALDI sample preparation: saturated 4-HCCA solution

Necessary reagents and instrumentation:
It is critical that all reagents are of the highest quality (e.g. HPLC grade).
  • Powder-free gloves (latex or nitrile)
  • Recrystallized a-cyano-4-hydroxycinnamic acid (4-HCCA)
  • Organic solvents: acetonitrile (ACN, A) or isopropanol (I)
  • Acids: trifluoroacetic acid (TFA, T) or formic acid (F)
  • Water (W)

It is very important to wear powder-free gloves during the preparation of the matrix solution.
  1. Choose solvents based on the hydrophobicity of the peptides and proteins to be analyzed (listed from hydrophilic to hydrophobic below). The FWI solution tends to favor high mass peptides (i.e. >4,000 u), and they also should be used for protein analysis. In contrast the other two 4-HCCA solutions (WA and TWA) favor low mass peptides.
    1. WA = 2 parts water, 1 part acetonitrile.
    2. TWA = 2 parts water, 1 part acetonitrile, 0.1% TFA (final concentration).
    3. FWI = 3 parts formic acid, 1 part water, 2 parts isopropanol (used for hydrophobic peptides and proteins, e.g. membrane proteins). Note that if the matrix is prepared in FWI solution, the peptides and proteins will become formylated at Ser and Thr residues within 30 minutes. Data acquisition should proceed immediately after sample spotting.

  2. Recrystallize 4-HCCA before preparation of the matrix solution.

  3. All reagent must be at room temparture. Recrystallized 4-HCCA must be recovered to room temperature as well. Use of cold reagents might cause the matrix to form clusters, which are very hard to redissolve.

  4. Start dissolving the matrix in the organic solvent.

  5. Scrape the inner walls of the vial with the pipette tip to dislodge the matrix powder from the walls and form a suspension.

  6. Vortex thoroughly.

  7. If applicable, add the acid and vortex thoroughly.

  8. Add water and vortex thoroughly.

  9. Pellet the excess matrix by centrifuging the vial for 6 minutes at 14000 rpm. The pellet can be reused, if stored at -20 °C.

  10. Transfer the supernatant to a fresh tube, taking care not to disturb the powder at the bottom.

  11. Once prepared, you may store the matrix solution in a microcentrifuge tube with the top wrapped in Parafilm® at room temperature in the dark (the matrix solution is typically good for 2-3 months).


S.L. Cohen, B.T. Chait "Influence of Matrix Solution Conditions on the MALDI-MS Analysis of Peptides and Proteins" Anal Chem 68 (1996) 31.

R.C. Beavis, B.T. Chait "Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Proteins" Methods in Enzymology. Vol. 270 (1996) Chap. 22, p. 519-551.

M. Cadene, B.T. Chait "A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins" Analytical Chemistry 72 (2000) 5655-5658.