Data independent acquisition methods for metabolomics

1. Data independent acquisition methods for metabolomics Stephen Tate, Ron Bonner AB SCIEX, 71 Four Valley Drive, Concord, ON, L4K 4V8 Canada MS/MS spectrum, 300 - 325 INTRODUCTION Data independent analysis collects MSMS Spectra of all ions without prior information of parent molecules SWATH™ MS subsets the MS range into windows which are then subjected to fragmentation providing increased selectivity Compatible with all types of sample introduction i.e. ion mobility, flow injection or LC Data is processed at the spectral level and utilizes sample information to identify differences cycle time MS/MS spectrum, 225 - 250 m/z MS/MS spectrum, 150 - 175 A high resolution product ion spectrum is acquired for every precursor mass within the quadrupole window, shown by the different arrows. This is repeated across the flow injection peak and the data from each isolation window is summed (signified by the colour bars) Spectral Peaks are identified in the summed product ion spectra for each isolation window. The variables are characterized by precursor window and fragment mass. The parent MS for the data is also available and this is also summed and used in the data processing. For each sample, the MS and MSMS spectra for each Swath can be processed together or separately using the MarkerView software and PCVG [1] MATERIALS AND METHODS Sample Preparation Urine samples were collected from a willing healthy person for a period for 30 days; no attempt was made to control behavior, diet etc., but on two occasions the subject visited the dentist and was treated with a rapid clearance anesthetic (Articaine, MW 270.10327). Mid stream collection occurred at the same time each day and samples were stored at -20°C ;prior to analysis the samples were diluted 1:5 in water formic acid and held at 4°C in the autosampler. MS/MS Conditions An AB Sciex 5600 TripleTOF™ system with Turbo V™ source was used. Samples (10ul) were introduced at a flow rate of 5 ul/min and the mass spectrometer was operated in SWATH [3] mode. Q1 was stepped in 25 amu windows from mass 100 to 1000 amu with the collision energy set dynamically for each window with a collision energy spread of 10eV applied to the central value. Data were acquired for 100 ms for each Swathgiving a total cycle time of ca. 4 sec. A TOF-MS experiment was also included in the scan set to allow accurate mass determination of the precursor ions. MS and Swath MSMS data are stored as separate experiments (one per swath) so processing and visualization can use them individually or in combinations. Data Processing The PeakView™ software was used for visual comparison. For statistical analysis data was extracted using a research version of the MarkerView™ software that extracts single spectra from FIA runs and can process experiments individually or all at once; variables are determined by applying a peak finder to the spectra. The MarkerView™ software was used for the analyses and the PCVG algorithm [4] used to find groups of correlated variables. Correlation across the samples allows related ions to be determined, in this case the fragment peak list spectrum of articaine. The spectra from the two SWATH’s are similar suggesting that one is a metabolite (actually the glucuronide with M+H+ at 447). Fragment prediction tools in the PeakView software help structural interpretation. • CONCLUSIONS • SWATH MS combined with flow injection analyses provides information rich data in a high throughput manner • The availability of MSMS data offers the potential to distinguish isobaric molecular ions with different fragment ions • Samples can be compared based on MS data, MSMS data from individual Swaths or a combination; processing individual Swaths reduces the total variance and makes analysis easier. • PCVG helps interpretation by finding groups of variables that are correlated across the samples which often correspond to molecular mass spectra • Correlation of MSMS across samples provide ions which are related and allows structural elucidation of the compound • REFERENCES • 1 Ivosev G, Burton L, Bonner R, Anal Chem 2008, 80, 4933 • TRADEMARKS/LICENSING • For Research Use Only. Not for use in diagnostic procedures. • The trademarks mentioned herein are the property of ABSciexPte. Ltd. or their respective owners. AB SCIEX™ is being used under license. Group 1 Group 13 In contrast, processing the MSMS data from a single SWATH (m/z 250 – 275) immediately reveals the dentist samples. PCVG provides the corresponding set of fragment ions because they are correlated across the samples. Other patterns can be seen in the data, for example, group 2 appears to correspond to samples collected on Saturdays. Group 2 Because of the high variability PCA of the Parent molecule intensity shows little or no structure. Manual examination of the groups assigned by PCVG revealed a set of variables that are present in the samples obtained following dentist visits and that included the M+H+ ion of articaine (m/z 271.11109).