CPSA Digest 2002

Proceedings -Wednesday, October 9, 2002

WeOE1

Desorption/Ionization on Silicon Mass Spectrometry (DIOS/MS) for High Throughput Lead Discovery and Optimization

Background
MALDI MS (matrix-assisted laser desorption/ionization mass spectrometry) is a fundamental technique for the identification of biomolecules. An analysis using MALDI typically dissolves analyte within an organic matrix, which vaporizes upon pulsed-laser radiation, carrying analyte with it. Direct desorption/ionization without any matrix has been studied using various surfaces.

Scientists at The Beckman Center for Chemical Sciences, a part of The Scripps Research Institute (TSRI) in La Jolla, CA, have developed a new ionization strategy for biomolecular mass spectrometry based on pulsed laser desorption/ionization from a porous silicon surface. Desorption/ionization on silicon (DIOS) uses porous silicon to trap analytes deposited on the surface; laser radiation vaporizes and ionizes the molecules without the presence of any matrix material. DIOS has been demonstrated for biomolecules at the femtomole and attomole levels with little or no fragmentation, in contrast to other direct desorption/ionization approaches.

Premise
DIOS-MS is presented as a unique scanning approach for the high throughput screening of enzyme activity and enzyme inhibitors. A MALDI mass spectrometer equipped with automated, multisampling capabilities was used to facilitate data collection and analysis. The DIOS chips were simply attached to the modified standard target plates using conductive carbon tape. Internal standards were added into analyte solutions for mass calibration and quantitation. Analytes were deposited directly onto the porous silicon surfaces and allowed to dry before DIOS-MS analysis. The sample plate was then loaded into the mass spectrometer and analyzed using a computer-controlled algorithm. Note that the only requirement for the successful implementation of this approach is that the substrate or product be ionizable.

DIOS Chips Specifications:
Currently DIOS chips are manufactured in two standard sizes: 0.75 x 0.75 in and 1.25 x 1.25 in. Standard DIOS chips have uniform or patterned micrometer-thick porous layer with nanocrystalline architecture generated on flat crystalline silicon. Thickness: 0.5mm. Spot diameter: 1mm, 100 (10 x 10) spots per chip.

Value of the Technology
DIOS-MS offers many unique advantages over traditional screening techniques (e.g., fluorescence which can yield false negatives and false positives):

• Good sensitivity
• Low background ion interference
• High salt tolerance
• No matrix required
• Performed with commercially available MALDI instruments
• Potential for incorporation with automation, microfluidics and microchip technology
• Simplified sample preparation
• Easily operated by a biochemist or mass spectrometrist
• Porous silicon surfaces can be reused repeatedly with only little degradation.

DIOS-MS is useful in many applications, such as:

• Proteomics (protein identification, protein functional characterization, kinetics and inhibition of protein functional activity)
• High-speed Proteomics
• Small molecules analysis (no low mass limit)
• Automated high throughput mass spectrometry

  Analysis
  The inhibitor screening of acetylcholinesterase, an enzyme which catalyzes the hydrolysis of acetylcholine to choline, was performed using quantitative DIOS-MS. Typically, the reaction between an enzyme solution and an excess of the acetylcholine substrate reaches completion within 15 min. The reaction can be stopped or significantly inhibited when an active enzyme inhibitor is present. To test the viability of this approach, a series of potential acetylcholine inhibitors were incubated with acetylcholinestrase and acetylcholine for 30 min. Acetylcholine/choline ratios of the incubation products were then measured by DIOS-MS to determine inhibitory activity. In cases where the potential inhibitor had no activity, acetylcholine is almost completely converted into choline and very low acetylcholine/choline ratios are detected by DIOS; in cases where the inhibitor is active, a high acetylcholine/choline ratio was observed (see Figure). The current throughput of such enzyme inhibitor screening is about every 5 sec per sample and requires about 8 min to screen 100 compounds.

  

  DIOS has also been implemented as a high throughput assay for phenylalanine hydroxylase (PheOH) activity which is associated with the disease phenylketonuria (PKU). PKU is an inherited, metabolic disorder that is caused by a deficiency of the enzyme PheOH which converts phenylalanine to tyrosine. A goal in developing such an assay is to screen thousands of proteins for the discovery of a proteolytically stable form of the PheOH enzyme that will be stable against degradation in the gastrointestinal system after oral administration. When an internal standard is used for each analyte of interest (deuterium-labeled L-Phe and L-Tyr , [2H5]-ring-Phe, [2H4]-ring-Tyr, respectively), it is possible to quantify the ratio of transformation of L-Phe to L-Tyr and calculate the specific activity of the protein by traditional methods. Once preliminary screening using this assay has identified positive clones, more detailed kinetic and stability studies (including Vmax and Km determinations, binding constants of substrates, proteolytic stability, pH dependence of activity, temperature-dependence of activity) can be performed.

  References and/or Links
  
  Mass Consortium Corporation website at www.masscons.com/
  
  John J. Thomas, Zhouxin Shen, John E. Crowell, M.G. Finn, and Gary Siuzdak, "Desorption/Ionization on Silicon (DIOS): A Diverse Mass Spectrometry Platform for Protein Characterization," Proceedings of the National Academy of Sciences, 98(9) (2001) 4932-4937.
  Full Text available at www.masscons.com/2001PNASDIOS.pdf
  
  Zhouxin Shen, John J. Thomas, Claudia Averbuj, Klas M. Broo, Mark Engelhard, John E. Crowell, M.G. Finn, and Gary Siuzdak, "Porous Silicon as a Versatile Platform for Laser Desorption/Ionization Mass Spectrometry" Analytical Chemistry 73(3) (2001) 612-619.
  Full Text available at www.masscons.com/2001AnalChemDIOS.pdf
  
  Jing Wei, Jillian Buriak, and Gary Siuzdak, "Desorption/Ionization Mass Spectrometry on Porous Silicon," Nature 399 (20 May 1999) 243-246.
  Full Text available at www.masscons.com/1999NatureDIOS.pdf

  
  
  

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