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STRAIN DIFFERENTIATION AND DETERMINATION OF CAPSID PROTEINS OF COXSACKIEVIRUS BY MALDI-MS
Citation:
Talley, J. M., J A. Shoemaker, AND G S. Fout. STRAIN DIFFERENTIATION AND DETERMINATION OF CAPSID PROTEINS OF COXSACKIEVIRUS BY MALDI-MS. Presented at American Society for Mass Spectrometry, Orlando, FL, June 02 - 06, 2002.
Impact/Purpose:
This particular task is comprised of 4 subtasks: 1.) Characterization of Potential Viral Biomarkers by Mass Spectrometry; 2.) Characterization of Parasites by Mass Spectrometric Techniques;
3.) Rapid Discrimination of Bacterial Indicators of Fecal Contamination and Bacterial Pathogens by Mass Spectrometric Techniques; and 4.) Investigation of Aeromonas Virulence Factors Using Mass Spectrometry.
The purpose of this research project is to use mass spectrometric techniques, such as electrospray ionization (ESI), capillary electrophoresis (CE) and matrix assisted laser desorption ionization (MALDI) mass spectrometry, to provide "protein mass fingerprinting" and protein sequencing information for viruses, bacteria and protozoa that cause waterborne disease. These protein mass fingerprinting libraries will be evaluated to determine whether mass spectrometric techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between infective / non-infective genus and strains of the selected microorganisms. The characteristic proteins identified by mass spectrometry as markers of infectivity/viability or strain differentiation can then be used to develop more sensitive microbiological drinking water methods.
Description:
Introduction: Contamination of viruses in water environments (rivers, lakes, sources of drinking water) is a new threat and serious health problem. Amongst organisms discharged from sewage septic systems is the coxsackievirus (single-stranded RNA virus). Differentiation between different strains of the coxsackievirus using conventional biological methods takes up to several months. The capsid protein sequences of several coxsackie strains are known and therefore amenable to rapid analysis by matrix-assisted laser desorption (MALDI) mass spectrometry (MS). This study represents the first investigation of the four capsid proteins of coxsackie B1, B3, B5 and B6 viruses by MALDI-M.
Methods and Instrumentation: Virus purification consisted of underlaying the viral sample with sucrose and pelleting the virus through the sucrose pad at 10 degrees C for two hours. The virus was layered atop a 7.5-45% sucrose gradient, centrifuged at 10 degrees C for 1.5 hours and fractionated. The virus was then layered atop a 20-45% CsCI gradient and centrifuged at 10 degrees C overnight. The virus was collected and desalted with a microcon-100 concentrate. The viral sample was reacted with 0.5% Trifluroacetic acid (TFA) in water and mixed with a matrix consisting of a 70/30 acetonitrile in 0.1% TFA water. Acid-resistant Lab Label Protection Tape was applied to the target and acted as a hydrophobic surface. MALDI-MS experiments were performed on a Bruker BIFLEXIII. Thermal relaxation of the excited matrix resulted in absorption of the capsid protein into the gas phase. All capsid proteins were run in the linear mode and detected in the first detector by their mass-to-charge ratio.
Preliminary Data: Reacting the virus with TFA disrupted the protein capsid and released the four viral proteins (VP1, VP2, VP3 and VP4). Preliminary results from the capsid protein of the coxsackie B6 virus show four peaks corresponding to the four viral proteins, m/z 31,549 (VP1), m/z 14,317 (VP2[H]2+), m/z 13,047 (VP3[H]2+) and m/z 7,528 (VP4). The four capsid proteins from the coxsackie B5 virus were also easily identified and correspond to VP1 (m/z 33,274), VP2[H]2+ (m/z 13,801), VP3[H]2+ (m/z 13,699) and VP4 (m/z 6,939). The results thus far show that the differentiation between strains of coxsackievirus can be achieved by MALDI-MS. The information we obtained will aid microbiologists in developing a more rapid method of identifying viruses in drinking water.