MALDI Target

A third possibility is that partial separation will occur on the MALDI target, such as during matrix crystallization,96 which can be distinguished by rastering the laser beam. 

There is a recent hybrid between AP-MALDI and ESI, matrix-assisted laser desorption electrospray ionization (MALDESI) [202], where species desorbed from a MALDI target are subjected to an electrospray before entering the mass spectrometer. The method is similar to ELDI except that the analyte is embedded in a matrix as in MALDI. 

The peptide mixture on the MALDI target can be exposed to a chemical derivatization to confirm the identity of a peptide by the mass shift associated with the sequence-specific derivatization. A large number of possible derivatization reactions can be combined with the MALDI-TOF analysis. Their usefulness depends critically on the kinetics of the derivatization reaction, whether the reaction is complete with small amounts of peptides and whether only one product is generated. A visible MALDI signal can be generated from low atomole of peptide present under the laser beam (Vorm et al., 1994), but these amounts are often not sufficient... 

Fig. 10.9. MALDI targets (a) Bruker Scout26 target with some sample preparations using different matrices and (b) Micromass target suitable for 96 samples as delivered from standard well plates, (b) By courtesy of Waters Corporation, MS Technologies, Manchester.

Fig. 10.18. AP-MALDI ion source with extended transfer capillary. Insets (a) the target holder can be equipped with a 64-spot MALDI target or (b) a 10 x 10-spot DIOS chip. Adapted fromRef. [142] by permission. John Wiley Sons, 2002.

In this pull-down assay, the enzymatic reaction is carried out completely in solution. Samples taken from the reaction mixture are then transferred to a SAM-modified MALDI target, on which the remaining substrate and the reaction product are selectively immobilized. Subsequent to the extraction of the analytes, the target is rinsed, treated with matrix, and MALDI-MS analysis is carried out. A major advantage of this assay scheme is that the inherent danger of negative influences on the reaction kinetics, which may be caused by immobilization of the substrate as in standard SAMDI-MS-based assay formats, is circumvented. Additionally, by selective extraction of the analytes of interest and removal of the other... 

MALDI target for analysis. All data points were collected into one data file for clarity and speed. An example of a MALDI target prepared by eluant spotting is shown in Fig. 11.8. Figure 11.9 shows a microsome time course for a typical analyte, internal standard, and analyte-intemal standard ratio. This example illustrates the necessity of the internal standard for quantification. Half-lives were calculated for all compounds from both data sets. The half-lives obtained by the two ionization methods are plotted in Fig. 11.10. A good correlation was obtained between the two techniques. 

There are several ways to coat a MALDI plate. One is two-layer overlayer [62,63] In this method, the first layer on the MALDI target plate comprises the densely packed matrix microcrystals formed by the quick solvent evaporation of a matrix solution in acetone or a mixture of acetonitrile and water at room temperature. A mixture solution containing both matrix and sample is then deposited onto the first layer of small crystals to form uniform analyte-matrix microcrystals. The overlayer method is reported to provide improved results, particularly for analyses of proteins and peptides [64-66],... 

For a few tissue samples, the matrix deposition can be performed manually. A matrix solution is sprayed onto the tissue section with a hand-held thin layer chromatography (TLC) sprayer or an artist airbrush. The reproducibility of manual matrix deposition is an issue. When the manual sprayer is used, the MALDI target plate with the tissue section is held vertically about 15-25 cm from the sprayer nozzle. It is recommended to spray multiple coats of matrix across the tissue section and each coating cycle consists of passing the sprayer two to hve times across the tissue section and allowing the tissue to dry for about 1-5 min. This process is usually repeated between 10 and 20 cycles. 

Scheme4.88 Microfluidic system for MALDI protein analysis, (a) automated sample pretreatment and injection (b) microreactor (c) microdispenser used to deposit sample into nanovials (d) shallow nanovials on the MALDI target plate and (e) automated MALDI-ToF-MS analysis. Reprinted with permission from [345]. Copyright 2000 American Chemical Society.

MALDI target plate (Applied Biosystems, Foster City, CA, Cat no. V700668 )... 

Clean a MALDI target plate carefully. Wash sequentially in ethanol, distilled ultra pure water, soap, ethanol, and distiller water. Dry and polish the plate with polish. 

Figure 4.4. Protein identification by MALDI-TOF. Steps in protein identification by MALDI-TOF are shown. Prior separation by gel or liquid chromatography to a single protein is needed prior to enzymatic digestion with trypsin. Digested peptides are spotted onto a MALDI target with an appropriate matrix that assists desorption of peptides upon activaton by laser. Peaks from the resulting mass spectrum represent peptide ions that can be searched in a database to match a theoretical tryptic digest from known proteins. The more proteins that are matched, the greater the statistical confidence in assignment of a protein identification.

RC-MS stems from the observation that mass spectra of peptides and proteins can be generated by laser activation of chromatographic surfaces placed on MALDI targets. The product application of RC-MS, called surface-enhanced laser desorption ionization-time of fiight (SELDI-TOF), refers to a mass spectrometry method for measuring native proteins adsorbed (retained) on various chemical or biochemical surfaces (Figure 4.11). Although the concept of laser desorption of intact proteins is well established, it has been commercially exploited only recently as a means... 

Figure 1 5 MALDI spectrum from a 2-D gel spot excised from a human proteomic study in which the corresponding spectrum of the cathepsin D precursor could be identified after using SMEC micropreparation sample preparation followed by elution and spotting onto the MALDI target plate and MALDI analysis. The peptide mass fingerprinting revealed the identity of the protein using the Mascot bioinformatic software and the Swissprot protein database. The ( ) indicates the peptide masses corresponding to the cathepsin D precursor, and (T) the trypsin peptide fragments that were used for internal mass calibration.

The use of mass spectrometry (MS) techniques to monitor SP reactions has recently become possible through the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry (137) after in situ cleavage of a small number of resin beads (138-140). Although the compound is cleaved from the resin, the cleavage happens directly onto the center of the MALDI target and the method can be considered on-bead. 

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