Impact of increased MS/MS sensitivity on the untargeted metabolomics workflow

Using Zeno IDA on the ZenoTOF 7600 system

Kranthi Chebrolu1, Jason Causon2, Shaokun Pang1, David Cox2, Christie Hunter1
1
SCIEX, USA; 2SCIEX, Canada

Abstract

Data dependent acquisition (DDA or IDA) is a widely used analytical workflow for untargeted metabolomics using mass spectrometry. High sensitivity, high resolution and fast acquisition rates are critical for obtaining very good metabolite identifications in complex matrices. Here, the advantages of Zeno MS/MS on the ZenoTOF 7600 system was explored using a rat urine sample set. The large increases in MS/MS sensitivity resulted in a 15% improvement in identified metabolites.

RUO-MKT-02-13174-A_f0

Introduction

Data dependent acquisition (DDA or IDA) is a widely used analytical workflow for untargeted metabolomics using mass spectrometry. Typically, DDA consists of two steps. In the first step, an MS survey scan is collected for the specified mass range, and this data can be later used for quantification. In the second step, MS/MS is performed on the most abundant features observed in the survey scan, according to user defined filtering criteria. Maximizing the number of features sent for MS/MS and then identifying those features with high confidence across different samples is paramount for an efficient and effective DDA workflow. 

The acquisition rates for MS/MS on QTOF systems are very high, often allowing this feature identification workflow to be performed in the same sample injection. However, higher acquisition rates could significantly lower MS/MS data quality due to lower accumulation times used, which could consequently impact the identification of the compounds during the library search step. Ideally, both spectral quality and resolution are maintained when acquiring MS/MS at very high rates.

Many orthogonal injection QTOF systems suffer from duty cycle losses (duty cycle typically between 5 to 25%) in the MS region where the continuous ion beam is pulsed into the orthogonal TOF region of the system. However, on the ZenoTOF 7600 system, implementation of the Zeno trap has improved the duty cycle in this region to ≥90% providing significant increases in MS/MS sensitivity while maintaining high spectral resolution.1 Here, the impact of this sensitivity increase on the ability to identify urine metabolites using a high MS/MS acquisition rate in a single injection Zeno IDA workflow was explored. 

Figure 1.  Large sensitivity gains observed in Zeno MS/MS resulted in higher library scores. Urine metabolites with library scores between 50 and 90 in the Zeno trap on data (green) were aligned with the Zeno trap off data (red). Library scores were typically higher in the Zeno trap on data, see Figure 4 for more details.

Key feature of ZenoTOF 7600 system for untargeted metabolomics

  • The ZenoTOF 7600 system acquires high resolution, high quality spectra at very fast acquisition rates (up to 133Hz), enabling a single injection workflow for untargeted metabolomics
  • The Zeno trap significantly increases MS/MS sensitivity on low abundant precursors during Zeno IDA
  • The number of MS/MS acquired with spectral quality above 95 has doubled when the Zeno trap is activated during DDA
  • Spectral quality increase provides an increase in library matches of 15%
  • Two untargeted workflows explored using SCIEX OS software for library searching and MarkerView software for statistical interpretation