Targeted metabolomics in rat urine using Zeno MS/MS

Analysis on the SCIEX ZenoTOF 7600 system

Kranthi Chebrolu1, Jason Causon2, Robert Di Lorenzo2, Christie Hunter1
1
SCIEX, USA; 2SCIEX, Canada

Abstract

Targeted metabolomic research requires assays with high sensitivity and specificity, to obtain high-quality, quantitative results in complex biological samples and to deliver biological insight. Here, the targeted MRMHR assay has been developed on the ZenoTOF 7600 system for a small panel of metabolites in urine, to explore the impact of Zeno MS/MS on quantitative quality. Zeno MS/MS provided a 13-fold average increase in MS/MS sensitivity,  providing both high-quality, full-scan MS/MS data for confident metabolite identification, providing higher sensitivity quantification, and enabling use of smaller sample volumes. The full workflow with software tools has been demonstrated.

RUO-MKT-02-13050-A_f0

Introduction

Often researchers in academic labs and those performing clinical trials are only given very small amounts of sample, and need to get the most information from the modest volume. This requirement drives the need for targeted assays with high sensitivity and specificity, to obtain high-quality, quantitative results in complex biological samples and to deliver biological insight. High-resolution accurate mass (HRMS) systems have excellent utility in untargeted metabolomics and compound identification in complex matrices because of their high specificity and full-scan MS/MS quality. However, HRMS instruments typically have not been the chosen instrument to perform accurate quantification of a targeted panel of analytes because of their lower sensitivity. In addition, some high resolution platforms cannot keep high resolution at the scan speeds required for fast analyses.

With the introduction of the SCIEX ZenoTOF 7600 system, scientists can now achieve quantitative results at high speeds with high mass accuracy. The core innovation on the  ZenoTOF 7600 system is the Zeno trap that when activated, provides significant improvements in duty cycle due to the optimization of ion transmission from the collision cell into the accelerator.1 This duty cycle improvement provides a substantial increase in MS/MS sensitivity and thus enables targeted high resolution workflows. Here, a small, targeted assay for metabolites in urine from a diabetic rat model was used to characterize the MRMHR workflow and the impact of Zeno MS/MS on the quality of quantitative results.

Urine samples from a Zucker diabetic rat model were obtained and a targeted assay for 13 metabolites was developed. Sensitivity between the Zeno trap on and Zeno trap off methods was compared and a significant gain in MS/MS signal was observed (for example: cAMP fragment in Figure 1 shows a ~12-fold improvement). In this technical note, the complete workflow from data acquisition through to statistical analysis of results is described.

Figure 1. Significant sensitivity gains in MS/MS. Comparison of extraction ion chromatograms (XICs) for cAMP fragments obtained from MS/MS collect with Zeno trap on (blue) vs. Zeno trap off (pink). Signal/noise ratio improved ~12.5 fold when using the Zeno trap. This XIC is from a 2 µL injection of diluted urine.  

Key features of the ZenoTOF 7600 system for targeted metabolomics

  • Significant increases in  MS/MS sensitivity due to Zeno trap, which delivers  ≥90%1 duty cycle on MS/MS fragments while still maintaining fast acquisition rates (up to 133 Hz)
  • MS/MS XIC peak area gains of ~13 fold with Zeno trap activated for high‑sensitivity quantification of detected metabolites
  • Flexibility of a QTOF instrument for additional workflow
  • SWATH acquisition, data dependent acquisition, electron activated dissociation (EAD)
  • Powerful data processing tools in SCIEX OS software for accurate quantification, followed by multivariate statistical analysis using MarkerView software

Â