Sample preparation in the MALDI analysis process

Biomolecules such as proteins, peptides, sugars, and large organic molecules such as polymers, dendrimers and other macro molecules can be analyzed using MALDI. Sample preparation for MALDI is very simple; however, it is one of the most crucial steps in the MALDI analysis process. MALDI is more tolerant to sample contaminants, but contaminants can seriously disturb incorporation of sample molecules with growing matrix crystals. This results in bad spots on the target plate, leading to low signal to noise ratio, resolution, and sensitivity.

Samples can be prepared in two different ways. One removes the contaminants before applying them on to the matrix and one removes the contaminants after the sample is spotted on to the target plate either before or after adding the matrix.

Miniaturized chromatographic set-up is used for the first approach, while some scientists have cleaned in-gel digest of proteins using reverse phase(RP) HPLC microcolumns packed with different types of RP-HPLC beads (1). Now tips are also packed with RP or ion-exchange resin to remove salts and detergents from protein mixtures, and their effectiveness is shown by their recent commercialization. Tips and columns effectively remove MALDI contaminants and give small volume of sample, this can result in possible highly sensitive MALDI analysis of the samples.

Purification of biological samples on target plates involves synthetic membranes or surfaces. Membranes go on top of the target plate and the biological samples are spotted on top of the membranes. Biological samples interact with the membrane through strong hydrophobic forces. This enables samples to remain on the membrane while the buffers and salts are washed away. Then the MALDI-matrix solution is added to the purified samples on the target plate, ready for analysis. Perfluorosulfonated ionomer films, polyethylene membranes, nonporous polyurethane membranes, and C8 and C18 extraction disks are examples of membranes that have been used successfully in the past for biological mixtures. Self-assembled monolayers (SAMs) of octadecyl mercaptan on gold-sputtered disposable MALDI probe tips have been used to concentrate the sample and to act as a purification device. However, it is unfeasible because it requires overnight sample incubation to fully concentrate at the probe tip.