Ion production in the MALDI source depends on the generation of a suitable composite material, consisting of the matrix and analyte. The prototypical MALDI recipe (i.e. dried-droplet method) is really very simple: a solution of the matrix compound is prepared and thoroughly mixed with analyte solution. A droplet of the mixture is then dried on the sample target, resulting in a solid deposit of analyte-doped matrix crystals. The dried sample spot is finally introduced into the mass spectrometer for laser desorption/ionization.
It is generally accepted that analyte molecules must be distributed throughout the crystals rather than being confined to their surface for efficient ion production to take place. In fact, an important part of sample preparation is to find a matrix material that will actually dry out of solution with analyte (i.e. protein molecules) in the resulting matrix crystals, rather than elsewhere in the deposit.
It is generally believed that conditions leading to the matrix isolation of analyte and the formation of small and homogeneous matrix/analyte co-crystals facilitates the overall ionization process, resulting in reduced matrix signal and stable and reproducible analyte ion yields.
Matrix isolation is easily accomplished by using a large molar excess of matrix to analyte (i.e. typically 100 to 10,000). Analyte and matrix concentrations in the microliter-sized droplets deposited on the metal MALDI target for evaporation are typically in the 1-10 uM and 1-10 mM range, respectively.
The formation of homogeneous analyte-matrix crystals is more elusive and has been the subject of several studies. Inhomogeneous crystallization slows down the data acquisition process as it forces the MALDI user to search for so called “sweet spots” on the crystal surface to obtain a stable ion signal. Inhomogeneous sample crystallization also affects the sample-to-sample reproducibility of MALDI results. These problems usually gets worse in samples with a low abundance of analyte or with high levels of contaminants.
Several different methods for growing homogeneous analyte-doped matrix crystals have been described in the mass spectrometry literature. The section titled “Crystallization Methods” on the previous page introduces some of the most widely used approaches, and describes the known pros and cons of the different sample crystallization techniques.