This study demonstrates the use of Agilent’s new MS/MS spectral metabolomics library to elucidate the identification of several amino acids that are important in malaria metabolism.
This document describes an untargeted metabolomics study using an Agilent LC/MS/MS Quadrupole Time-of-flight (A-TOF) system to analyze metabolites in Plasmodium falciparum infected and noninfected human red blood cell extracts. The study highlights the challenges of compound identification in metabolomics and the necessity of high-confidence identification for biochemical pathway analysis.
The authors utilized the Agilent METLIN Personal Compound Database and Library (PCDL), which contains over 2,200 standards, to confirm the identity of specific amino acids, including arginine, ornithine, and citrulline. The document details the specific LC and MS/MS acquisition conditions used, the data processing workflow involving MassHunter Qualitative and Mass Profiler Professional software, and the results of library matching using reverse and forward search algorithms to validate compound identities.
| Collision cell energy | 10 and 20 eV |
|---|---|
| Column temperature | 60°C |
| Injection volume | 10 ul |
| Autosampler temperature | 4c |
| Flow rate | 0.6 mL/min |
| Drying gas temperature | 325°C |
| Drying gas flow | 9 L/min |
| Nebulizer pressure | 45 psig |
| Capillary voltage (positive mode) | 4000 V |
| Fragmentor voltage | 140 V |
| Skimmer voltage | 65V |
| OCTIRFVpp | 750 V |
| Isolation width | ~ 1.3 m/z |
| Instrument mass range | 1700 Da |
| Acquisition rate | 3.35 spectra/s |
| TOF spectra mass range | 25 to 1000 m/z |
| Precursor ion tolerance | +/- 10 ppm +/- 2 mDa |
| Product ion tolerance | +/- 35 ppm +/- 2 mDa |
| Library match threshold (reverse) | Above 80 |
| Library match threshold (forward) | Below 50 is uncertain |
The Find by Molecular Feature (MFE) algorithm in Agilent MassHunter Qualitative B.04 software was used.
The library contains MS/MS spectra for over 2,200 compounds.
The collision cell energy was set to 10 and 20 eV in positive ion mode.
A reverse search score above 80 is considered a good match.