Sample preparation strategies in MALDI

Since the method inception in 1980-ies, peptides containing R (arginine) or L (lysine) somewhere in their sequences, e.g. tryptic peptides, were the initial targets for MALDI MS analysis¹. At its initial stage, MALDI MS technique was used mostly for peptide fingerprinting²,³. In the following decades, MALDI analysis was applied to characterization of proteins and proteins complexes, small molecules, carbohydrates, and lipids.

Although a large number of MALDI matrices have been identified over the years, most researchers rely on a relatively small set of established compounds. Alpha-Cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB) are three widely used matrices. These matrices are particularly suitable for the analysis of peptides (CHCA/DHB) and/or proteins (DHB/SA); DHB is also widely employed for the analysis of carbohydrates and lipids. A key concept behind obtaining a reasonably good signal in MALDI analysis of peptides is a match of hydrophobicity of peptides under analysis and that of the matrix⁴. So for instance, peptides containing tyrosine (Y) and/or tryptophan (W) amino acid residues are more hydrophobic than those with Aspartic (D) or Glutamic amino acids (E). Accordingly, CHCA is considered as mostly hydrophobic matrix whereas DHB as hydrophilic⁵.

Another example of matrices that is particularly suitable for the analysis of polar lipids, for example triacylglycerides, are 2,4,6-trihydroxyacetophenone (THAP) and 2,5-dihydroxyacetophenone DHAP; these two matrices are also an example of a less acidic, noncarboxylic acid matrices.

More “specialized” matrices that can enhance specific features of the MALDI MS analysis (e.g., the in-source decay) or enable the analysis of specific types of analytes. For example, because of its almost “neutral” pH, 6-aza-2-thiothymine (ATT) is particularly well suited to analysis of non-covalently bound complexes, and 1,5-diaminonaphthalene (DAN) is a matrix with which high in source decay yields are achieved. Recently, 3-hydroxy-4-nitrobenzoic acid ⁶ has been used as MALDI matrix improving peptide sequence coverage using the in-source decay. It was shown that addition of ammonium sulfate could also increase the rate of the in-source decay ⁷.

The most efficient matrix for mass analysis of peptides with MW\<3 kDa or protein tryptic digests was found to be CHCA. Sometimes, ammonium salts⁸ or serine⁹ are added to the matrix solution to reduce the in-source decay or diminish the abundance of sodium or potassium adducts.

In most MALDI applications, especially those aimed at quantitative mass analysis, it is very important to observe a uniform MS response when scanning across MALDI sample with focused laser beam. So it is vital to form a sample spot containing similarly-sized small crystals (with characteristic size of several um) produced after evaporation of a solution where matrix was mixed with peptides. The sample deposition method where a solution containing a mixture of analytes and matrix is deposited onto a target plate and then slowly evaporates forming a field of microcrystals is referred as the dried-droplet method.

CHCA is known for producing a field of small (10-15 um) close in size crystals after solution containing CHCA evaporates.

“Classic” CHCA sample preparation method for peptide mass analysis

In recently developed sample preparation method that gives a group of tightly spaced small crystals of approximately same size (Sigma-Aldrich, see Product Information for ProteoMass MALDI Calibration kit, Catalog MSCAL-4), first the solution of CHCA at ~8 mg/mL concentration is prepared mixing three solvents:

• 13% of Ethanol
• 84% of Acetonitrile (AcN)
• 3% of Water containing 0.1% TFA

Up to 10 mg of CHCA matrix is added to 1 mL of Ethanol/ACN/Water solution and well mixed at room temperature using the vortex mixer (e.g., Vortex Genie 2, Scientific Industries Inc, Bohemia NY ).

Then one needs to fix the vial vertically and wait for approximately 15 min. After that, ~ 500 uL of “supernatant” is slowly drawn from the top layer of the solution in the 1 mL vial and discharged into a separate non-transparent (e.g., brown colored) vial. Matrix solution can be stored for a few days in a refrigerator where temperature is maintained at 2-3 degree C. It is advised to cover the bench with the vials with Aluminum foil to prevent the matrix solution from being exposed to light. The 3-day old matrix solution should be replaced with a fresh solution.

Peptide standards from MALDI calibration mixture (e.g., ProteoMass™ MALDI Calibration Kit from Sigma-Aldrich) to be used for the mass analysis and evaluation of a limit of detection (LOD) are dissolved in 80%/20% (Water/AcN) solution to concentrations of 1-100 uM. Then 1 or 10 uL aliquots of peptide solution are added to 10 uL or 100 uL, correspondingly, of CHCA solution. One is advised to wait for 20-30 min without using the lab mixer for the peptide and CHCA solutions to be fully mixed. So the resulting solution is rich in AcN and Ethanol.

Finally, 1-uL aliquots of the combined (CHCA + peptide) solution are deposited on MALDI target (mostly a stainless steel plate or golden plated microscopic glass slide) at room temperature. If one looks at a fresh dried spot using optical microscope with 40x magnification, one finds a field of small crystals, approximately of the same size. The produced sample spot is typically ~1.5 mm in diameter while the average crustal size is 15-20 micron.