Application of DIOS in Bioanalysis

Desorption/ionization on porous silicon has been successfully applied to the direct mass analysis of a variety of analytes and analyte mixtures, e.g. from exocrine tissues as well as from single neurons. In forensic analysis, DIOS-MS was applied for the analysis of small molecular weight polymers from biological samples, e.g. spermicides or polyethylene glycol polymers. But it has also been applied in the fields of drug discovery [17] or fatty acid analysis [18]. DIOS-MS has early been used for the monitoring of enzymatic reactions, too. In 2001, Thomas et al. investigated a multi-enzyme system comprising of a glucosidase (mannosi-dase II), a lipase (phospholipase A2) and an esterase (acetylcholinesterase, AChE) and their respective substrates [19]. All reactions were carried out directly on the silicon surface. Subsequent to quenching, which was accomplished by the evaporation of the solvent, direct DIOS-MS analysis of the dried reaction mixture was performed. AChE was reacted with its substrate acetylcholine, and deuterated choline was used as an internal standard for quantification (Fig. 8.8).

A time-resolved reaction profile was generated by plotting the choline formation vs time, and inhibition of acetylcholinesterase by three different inhibitors was studied in three independent reactions on a single target plate, thus allowing screening the inhibitory activities within 15 min, including sample preparation time. In order to assay mannosidase II, an oxidized silicon surface was used on which best signal responses of the carbohydrate analytes were obtained. The possibility to tailor the silicon's surface properties is thus one of the major advantages of DIOS-MS analysis when different reaction mixtures are concerned. The activity of phospholipase A2 was determined by reacting the enzyme with a tria-cylglycerol phospholipid, which yielded a lysophospholipid species upon enzymatic conversion (Fig. 8.9). After 30 min of incubation directly on the DIOS chip, the product species could be monitored in the MS as their sodium and potassium adducts.

A further advantage, as described by Thomas et al. [19], is the possibility of protein identification that follows the functional characterization of the enzyme. The activity of an enzyme is initially determined by following the substrate consumption and product formation in the first assay (Fig. 8.10). Since no matrix components are present in the sample spot, the immobilized enzyme is then directly

Fig. 8.9 The activity of phospholipase A2 can be determined by following the consumption of the triacylglycerol phospholipid and the formation of the lysophospholipid.

digested with site-specific proteases. Afterwards, the digest is analyzed again by means of DIOS-MS and the protein fragments generated can be used for the correct identification of the protein.

An automated DIOS-MS-based approach as screening assay for enzymatic activities and enzyme inhibitors was published in 2004 [20]. A DIOS-MS platereader assay was employed in an enzyme activity screening, searching for new enzymes with activity similar to phenylalanine hydroxylase (PAH). Determination of kinetic parameters as a measure of catalytic activities was carried out by varying the substrate concentration and monitoring product formation as well as substrate consumption. For quantification, deuterium-labelled phenylalanine and tyrosine as internal standards were used. In a second set of experiments, the DIOS-MS plate reader assay was employed in a screening for potential inhibitors of acetylcholinesterase. The library of potential inhibitors comprised more than 900 compounds, including some known reference inhibitors. All enzymatic reactions in the study were carried out offline and aliquots of the reaction mixtures were spotted onto the DIOS target. Thus, speed and precision of sample deposition become the most crucial point for the application of this system. Employing

Fig. 8.10 Sequential functional characterization and structural identification of an enzyme. Initially, information about the activity is obtained by assessing substrate consumption and product formation. Afterwards, the enzyme is digested on the plate, and the formed peptide fragments (Fn —F4) are determined by means of mass spectrometry.

Fig. 8.10 Sequential functional characterization and structural identification of an enzyme. Initially, information about the activity is obtained by assessing substrate consumption and product formation. Afterwards, the enzyme is digested on the plate, and the formed peptide fragments (Fn —F4) are determined by means of mass spectrometry.

an electrospray deposition device (Fig. 8.7), sample homogeneity could be significantly improved. With this approach, the high potential of DIOS-MS-based assays as a tool in high-throughput screening for either determining enzymatic activities or detecting potential inhibitors has been clearly demonstrated. Nevertheless, it should be noted that the number of different enzymatic systems studied by this interesting technique is still rather small and the exact mechanism of the ionization by DIOS is not yet fully understood.

The surface of the porous silicon chip offers multiple possibilities for the covalent coupling of functional groups. Thus, functionalization of a DIOS target by immobilization of trypsin was described by Xu et al. [21]. The enzyme was immobilized using cyanuric chloride as coupling agent following an amino-functionalization (Fig. 8.11).

It could be shown that the immobilized enzyme retained its bioactivity and the kinetic parameters for the trypsin-catalyzed proteolysis of an appropriate substrate were determined. However, the value for vmax was found to be lower than for free trypsin, thus indicating a slight loss of activity likely to be related to the immobilization of the enzyme. The trypsin-functionalized DIOS target was used for peptide-mapping analysis of two model proteins: cytochrome c and bovine serum albumin (BSA) were incubated on the target and after evaporation to dryness directly analyzed by means of DIOS-MS. The signal intensity of the peptide fragments generated was found to be low, which might be due to the surface modification. Therefore, a small amount of a typical MALDI matrix (a-cyano-4-

Fig. 8.11 Covalent coupling of trypsin to a silicon surface. In a first step, free silanol groups are reacted with 3-amino-propyltriethoxysilane. The amino-functionalized surface is then treated with 2,4,6-trichloro-1,3,5-triazine (cyanuric chlorid). Finally, trypsin is covalently bound via a free amino group of the protein.

Fig. 8.11 Covalent coupling of trypsin to a silicon surface. In a first step, free silanol groups are reacted with 3-amino-propyltriethoxysilane. The amino-functionalized surface is then treated with 2,4,6-trichloro-1,3,5-triazine (cyanuric chlorid). Finally, trypsin is covalently bound via a free amino group of the protein.

hydroxycinnamic acid) was added to increase the signal response significantly. In the presence of matrix, 19 peptide fragments could be assigned for cytochrome c and 54 peptide fragments for BSA. This means that the DIOS-MS approach can be ''converted'' into a MALDI-MS scheme for enhancing signal intensities, when needed.

Was this article helpful?

0 0

Post a comment