Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Improved Metabolite Identification in a Single Injection with Data Independent Acquisition for Untargeted Metabolomics Workflow (#599)

Arianna Jones 1 , Bob Proos 1
  1. SCIEX, Framingham, MA, United States

Comprehensive metabolite identification with MS/MS library spectral matching can be problematic for data dependent acquisition (DDA) workflows as it often requires multiple injections for each sample. SWATH® Acquisition, a Data Independent Acquisition (DIA) method, with optimized variable windows, provides a powerful workflow requiring only a single injection per sample for each polarity.  In addition to capturing product ion spectra for all detectable analytes, SWATH Acquisition also provides the option of quantitation at either the MS or MS/MS level allowing a comprehensive qualitative and quantitative analysis of metabolites in complex biological samples like plasma. The speed of the X500R QTOF  and the TripleTOF® 6600 QTOF system allows for the top 20 selection in DDA or 20 variable SWATH acquisition windows in DIA analysis with a cycle time of 651 msec for both the DDA and SWATH Acquisition methods, which provides more than 9 points across a peak of 6 seconds providing accurate and reproducible integration.

Human Plasma, NIST SRM1950, was purchased from NIST and metabolites were extracted using cold methanol.  Both the DDA and SWATH Acquisition methods compared in this study were single injection workflows, using the same simple RP chromatography with a 20 minute run time per injection.  Data was processed using SCIEX OS software for non-targeted peak finding with spectral library matching.

Top 20 DDA method was able to identify 476 of the features on the basis of precursor mass and MS/MS spectral matching.  The 20 variable window SWATH acquisition method resulted in an additional 215 features being identified, a 45% increase, for a total of 671 compounds identified in a single injection.  This study has shown that variable window SWATH acquisition can be a useful for improving compound identification in untargeted metabolomics.