Laser desorption MS for biomolecules Direct laser desorption MS is tough on molecules. The use of matrixes in MALDI makes laser desorption gentler but introduces its own drawbacks, such as background ions in the mass spectrum. Gary Siuzdak and Jing Wei of The Scripps Research Institute and Jillian M. Buriak of Purdue University have found a way to perform biomolecular laser desorption MS that doesn't need a matrix. They call their method desorption/ionization on silicon (DIOS) MS. Instead of using a matrix, the researchers placed samples on a porous silicon surface. They prepared the surface from flat crystalline silicon by galvanostatic etching. The properties of the porous layer—generally several micrometers thick—could be controlled by the choice of the silicon wafer and the etching conditions. The stability of the surfaces allows them to be used repeatedly. The authors used the surfaces for DIOS-MS of compounds that ranged in size from 150 to 12,000 Da, including carbohydrates, peptides, glycolipids, natural products, and small drug molecules. Compounds were dissolved in aqueous solutions and deposited on the silicon surfaces.
Unlike MALDI, this method does not suffer from matrix interference when used for small molecules. Peptides generated good signals at levels as low as 700 amol and could be analyzed even in a saturated salt solution. The pores in the silicon appear to be necessary to perform these measurements. In control experiments, the authors tried to no avail to obtain mass spectra of compounds on glass, air-oxidized single-crystal silicon, silica-gel thinlayer chromatography plates, and gold MALDI plates. They also found that smaller pores generated more intense signals. To better understand the mechanism, they used the DIOS approach with four surface modifications hydride (the native surface) dodecyl ethyl phenyl and oxide The hydrophobic surfaces generated the more intense signals
Experimental configuration for the DIOS-MS experiments, ,a) Four porous silicon plates on a MALDI plate, (b) The silicon-based laser desorption/ionization process, (c) Cross section of porous sillcon and the surface functionalities after hydrosilylation; R represents phenyl or alkyl chains. (Adapted with permission. Copyright 1999 Macmillan Magazines.)
Probing protein secondary structure Because of the complexity of proteinfolding dynamics, it is impossible to predict secondary and tertiary structures based on primary protein sequences. Protein structures evolve over several different time scales, from nanoseconds for the nucleation of a-helix and (3-sheet secondary structures to milliseconds for the formation of tertiary structures. Fluorescence and IR spectroscopy have recently been used to probe protein folding in nanosecond time scales. Sanford A Asher and coworkers at the University of Pittsburgh take it one step further using nanosecond transient UV resonance Raman spectroscopy (UVRS) to examine the earliest stages of a-helix secondarystructure evolution The researchers investigated the thermal unfolding of the a-helical peptide A5[AAARA]3A (AP). UVRS excitation in the 200-nm region selectively
The authors suggest that DIOS-MS could be used for a broad range of applications. For example, the surface could be derivatized with receptors and used to identify ligands. Existing MALDI mass spectrometers can easily be converted to DIOS by altering the MALDI sample plate. In addition, porous silicon can easily be integrated in chip-based microfluidic chemical reactors. (Nature 1 9 9 9 , 399, 243-46)
enhanced amide vibrations, which depend on secondary structure, and thus can be used to selectively probe protein secondary structure. Static 204-nm UVRS spectra of AP were obtained at several different temperatures, ranging from 4 to 70 °C. As the temperature increased, the a-helical content decreased, indicating that at high-temperatures AP is primarily a random coil. This result was consistent with circular dichroism data. A transient UVRS spectrum of AP was then obtained 95 ns after a temperature jump from 4 to 69 °C. The resulting UVRS changes were consistent with those expected due to the partial unfolding of a-helical AP to a random coil. Transient UVRS spectra of AP were allo obtained after different delay times following a temperature jump from 4 to 33 ° °. The static UVRS spectrum obtained at 4 °C waa subtracted from each of the transient spectra, yielding transient difference spectra. In
addition, a static "infinite" spectrum, which represents the difference spectrum at an infinite delay time, was obtained by the difference between static UVRS spectra measured at 37 °C and 4 °C. UVRS changes were found to depend on the probe delay time. Random coil formation was observed only at longer delay times. Assuming monoexponential kinetics, the authors calculated relaxation times for various temperature jumps, which were then used to calculate folding- and unfolding-rate constants and activation barriers. The temperature dependence of the a-helix unfoldingrate constant showed Arrhenius behavior. In contrast, the folding-rate constant showed a negative activation barrier. The authors attribute the slower folding kinetics and their unusual temperature dependence to a bottleneck, which may possibly involve a-helix nucleation. (J. Am. Chem. Soc. 1 9 1 9 121 4076-77)
Analytical Chemistry News & Features, July 1, 1999 4 3 9 A
