Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) is a novel method for generating and analyzing gas-phase ions that employs direct laser vaporization. The structure and physicochemical properties of the porous silicon surfaces are crucial to DIOS-MS performance and are controlled by the selection of silicon and the electrochemical etching conditions. Porous silicon generation and DIOS signals were examined as a function of silicon crystal orientation, resistivity, etching solution, etching current density, etching time, and irradiation. Pre-and postetching conditions were also examined for their effect on DIOS signal as were chemical modifications to examine stability with respect to surface oxidation. Pore size and other physical characteristics were examined by scanning electron microscopy and Fourier transform infrared spectroscopy, and correlated with DIOS-MS signal. Porous silicon surfaces optimized for DIOS response were examined for their applicability to quantitative analysis, organic reaction monitoring, post-source decay mass spectrometry, and chromatography.

Since the advent of matrix-assisted laser desorption͞ionization and electrospray ionization, mass spectrometry has played an increasingly important role in protein functional characterization, identification, and structural analysis. Expanding this role, desorption͞ionization on silicon (DIOS) is a new approach that allows for the analysis of proteins and related small molecules. Despite the absence of matrix, DIOS-MS yields little or no fragmentation and is relatively tolerant of moderate amounts of contaminants commonly found in biological samples. Here, functional assays were performed on an esterase, a glycosidase, a lipase, as well as exoand endoproteases by using enzyme-specific substrates. Enzyme activity also was monitored in the presence of inhibitors, successfully demonstrating the ability of DIOS to be used as an inhibitor screen. Because DIOS is a matrix-free desorption technique, it also can be used as a platform for multiple analyses to be performed on the same protein. This unique advantage was demonstrated with acetylcholine esterase for qualitative and quantitative characterization and also by its subsequent identification directly from the DIOS platform.M ass spectrometry is quickly becoming an essential tool for characterizing protein function, substrate specificity, and protein identity (1-5) as it complements or, in some cases, supersedes the utility of traditional biological methods (6, 7). For some of the most important proteomics applications, the high sensitivity and accuracy provided by modern mass spectrometry allow for unequivocal characterization and quantitative analysis of proteins and their chemical products (5,8,9). Ionization methods such as electrospray ionization used in liquid chromatography͞MS and matrix-assisted laser desorption͞ionization (MALDI) are the core innovations that allow for mass spectrometry to be used in protein characterization as well as in the determination of protein structure͞function relationships (10, 11). MALDI mass spectrometry has been particularly effective as a proteomics tool because of its relatively high tolerance of mixtures and biological contaminants (12, 13); however, its matrix requirement represents a limitation in interference in the low-mass region, preparation time, and the potential to perform sample manipulation after mass analysis. In addition, a prevailing obstacle toward protein characterization by using both MALDI and liquid chromatography͞MS is the loss of analyte material during protein separations, chromatographic separations, or functional studies that require the transfer of the sample for subsequent identification. One way to overcome these obstacles would be to both identify and functionally characterize proteins on a single surface.Desorption͞ionization on porous silicon (DIOS), a new method for the generation of intact gas phase ions (14), uses UV laser light to desorb intact analytes from the surface without matrix assistance. The procedure for producing DIOS surfaces involves a simple galvanostatic etching procedure (15...

The adsorption and reaction of tetraethoxysilane (TEOS) with hydroxylated SiO2 has been studied over the range of 100–450 K using transmission infrared spectroscopy. At 100 K, TEOS [Si(OC2H5)4] condenses on SiO2. Upon warming in vacuum, some of the condensed phase TEOS desorbs molecularly while a significant portion of the layer enters into a physisorption state. The physisorption state maximizes near 250 K, with strictly molecular desorption occurring upon warming to higher temperatures. When exposure occurs at 450 K, Si(OC2H5)4 reacts to form adsorbed siloxanes, thought to be a mixture of (SiO)2Si(OC2H5)2 and (SiO)Si(OC2H5)3. The adsorbed di- and triethoxysiloxanes decompose completely on heating in vacuum to 900 K. The chemistry of TEOS on SiO2 has been modeled using ethanol adsorption. Exposure of SiO2 to ethanol at 450 K leads to the formation of an adsorbed ethoxide species. Ethanol is shown to spectroscopically and chemically model the surface bound siloxanes produced upon reaction of Si(OC2H5)4 with hydroxylated SiO2 at 450 K. The vibrational spectrum of adsorbed ethoxide (SiOC2H5) is very similar to that of the adsorbed siloxanes produced from the adsorption of Si(OC2H5)4. The ethoxy modes are assigned through comparison of C2H5OH and CH3CD2OH adsorption. The temperature dependence of the decomposition of the adsorbed ethoxide is similar to that of the TEOS derived siloxanes. Decomposition of the adsorbed siloxanes is shown to evolve primarily ethylene.

A surface-based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS-MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS-MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS-MS system was also used as a high-throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface-based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications.

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