Our research methodology is based on following steps.
Total 5000 compounds will be requested from the Molecular Bank situated at the International Center for Chemical and Biological Sciences, University of Karachi. Other natural products will be purchased from commercial sources.
Working solutions of compounds will be prepared by dissolving compounds in 1:1 solution of water with 0.1 % formic acid and acetonitrile for positive mode analyses while water with 4 milli-molar ammonium acetate and acetonitrile for negative mode analyses. The final concentration is less than 1 ppm. An internal standard will be used in the solutions. Solvents used in this study will be of HPLC grade while deionized water will be obtained from MIlli-Q water assembly.
Compounds will be analyzed on positive ion mode by collision-induced dissociation (CID) electrospray ionization (ESI) on the QSTAR XL hybrid LC/MS/MS system (Applied Biosystems/MDS Sciex, Darmstadt, Germany). High-purity nitrogen gass will be employed as the collision and curtain gas delivered from nitrogen generator of Peak Scientific. The ESI interface parameters will be as followed: curtain gas flow rate 20 L/min, declustering potential DP1 60V, DP2 15V, nebulizer gas flow rate 30 L/min, focusing potential of 265V and ion spray capillary voltage of 5500 V. MS/MS spectra will be run at collision energies in the range of 20 to 55 eV. Internal Calibration method will be used for calibration. Harvard syringe pump (Holliston, MA) will be used for the introduction of samples into the mass spectrometer with 5 µL/min flow rate. MS2 experiment will be conducted by selecting the product ion.
Compounds (0.2 ng/mL trichloric acid as an internal standard) will be analyzed on negative ion mode by collision-induced dissociation (CID) electrospray ionization (ESI) on the QSTAR XL hybrid LC/MS/MS system (Applied Biosystems/MDS Sciex, Darmstadt, Germany). High-purity nitrogen gass will be employed as the collision and curtain gas delivered from nitrogen generator of Peak Scientific. The ESI interface parameters will be as followed: curtain gas flow rate 20 L/min, declustering potential DP1 -55V, DP2 -15V, nebulizer gas flow rate 25 L/min, focusing potential of -265V and ion spray capillary voltage of -4200 V. MS/MS spectra will be run at collision energies in the range of 20 to 55 eV. Internal Calibration method will be used for calibration. Harvard syringe pump (Holliston, MA) will be used for the introduction of samples into the mass spectrometer with 5 µL/min flow rate. MS2 experiment will be conducted by selecting the product ion.
Mass spectral database will be developed by using MassBank software and record editor (www.massbank.jp). This software is named as the MassBank Development Kit which is freely available at http://sourceforge.net/p/massbank/MDK/ci/master/tree/. MassBank has kindly agreed to support and train our staff members in one of their designated laboratories. The supporting email from Prof. Takaaki Nishioka (Nara Institute of Science and Technology, Graduate School of Information Science) is attached as Annexure V. These laboratories will support our research staff members for a period of 3-4 months and will provide necessary training for conversion of mass spectral data into the MassBank database format. The software package contains necessary tools for record generation and their incorporation into the database.
MassBank is an open-access project where the spectral data is open to every researcher in the world. However, it is not mandatory for the researchers who participate in the mass bank consortium to upload their data to the MassBank server. The data can be treated as in-house data and kept under the institutional policies. The following parameters will be carefully recorded for the database development.
Molecular weight of each compound will be recorded in positive and negative ion modes in high resolution
Different parameters of the analyzed compounds particularly collision energy will be carefully optimized for getting fragments with maximum intensity.
The fragmentation data of each compound will be recorded on its optimized collision energy
Database will be developed by pooling all the data in the form of separate database files with the names of the compounds. Software will be modified as per our requirement and will be integrated with the developed database and will provide searching of various spectra in high-throughput manner.
Validation of the developed database will be carried out by the re-analyses of compounds as unknowns and the recorded information will be searched with the developed spectral database to check the efficacy of the search algorithm and also to ascertain that the search algorithm does not give any false positives/ negative results.