Rapid screening methods for yeast sub‐metabolome analysis with a high‐resolution ion mobility quadrupole time‐of‐flight mass spectrometer

Teresa Mairinger,Stephan Hann,Ruwan Kurulugama,George Stafford,Tim J Causon,John Fjeldsted

doi:10.1002/rcm.8420

Abstract

RationaleThe wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front‐end separation techniques combined with high‐resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high‐throughput analyses.MethodsPrior to low‐field drift tube ion mobility (IM) separation and quadrupole time‐of‐flight mass spectrometry (QTOFMS) detection, rapid ultrahigh‐performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data‐independent all‐ions acquisition and a quadrupole broad band isolation (Q‐BBI) directed by IM drift separation.ResultsFor Q‐BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision‐induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 μM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM‐TOFMS‐based study.ConclusionsChimeric mass spectra, inherent to data‐independent analysis approaches, are reduced when using a drift time directed Q‐BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front‐end separation, a powerful approach for metabolite screening.

Highlights

Studies involving metabolomics rely strongly on the use of mass spectrometry technology in order to both confirm metabolite identity and quantify the small molecules in complex matrices ideally with minimal user intervention
Prior to low‐field drift tube ion mobility (IM) separation and quadrupole time‐of‐flight mass spectrometry (QTOFMS) detection, rapid ultrahigh‐performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract
We demonstrate that using advanced IM‐QTOFMS instrumentation equipped with a prototype continuous band quadrupole driver, the transmission settings and/or collision energy applied to dansylated precursors can be successfully directed by IM separation

Summary

Introduction

Studies involving metabolomics rely strongly on the use of mass spectrometry technology in order to both confirm metabolite identity and quantify the small molecules in complex matrices ideally with minimal user intervention. Obtaining a high‐resolution mass spectrum for an unknown metabolite is known not to be sufficient to enable confirmation of metabolite identity since, even if the chemical sum formula determination according to accurate mass and isotope pattern matching is accurate, the number of potential structure matches remains too high.[1,2,3] Because of these major challenges faced for non‐targeted metabolome assessment, employment of additional analytical selectivity via the use of high‐resolution fragment (HR MS/MS) spectra can be used to support metabolite identification as a complementary and chemically informative descriptor.[4,5] In the case of HR MS/MS, data‐dependent acquisition strategies rely on isolation and fragmentation of precursor ions which have exceeded a user‐defined intensity threshold or any other measurable criteria such as isotopologue pattern, mass defect or the presence of a diagnostic ion.[6] some limitations for non‐targeted assessment are apparent. These windows can be defined by the user according to both retention time and m/z considerations, which has proven extremely effective for improving proteome coverage despite challenges arising from chimeric MS/MS spectra

Results

Discussion

Conclusion