Presentation on theme: "High Performance MALDI MS, MS/MS, and Multiplexed MS/MS Tissue Imaging"— Presentation transcript:
1High Performance MALDI MS, MS/MS, and Multiplexed MS/MS Tissue Imaging Boone M. Prentice1, Kevin M. Hayden2, Marvin L. Vestal2, Richard M. Caprioli11Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, TN 372352SimulTOF Systems, Sudbury, MA 01776MSACL 2014 USMarch 1 - 5, 2014, San Diego, CA
2Why Imaging Mass Spectrometry? IMS combines molecular specificity with location704 m/z726 m/z733 m/z742 m/z761 m/z783 m/z787 m/z797 m/z809 m/z811m/z821m/z773 m/z805 m/z849 m/zmedullaRat KidneycortexThe multiplexed nature of MS analysis allows for the parallel acquisition of many different molecular signals, each which can be reconstructed to give a molecular picture.
3Imaging Mass Spectrometry Lipid & Protein Ion Images…………MatrixapplicationMatrix-Assisted Laser Desorption/IonizationLaserImages for individual m/zvalues integrated over all pixelsMS spectrum for each x, y coordinate (pixel)Sample PreparationSA – proteins/peptidesDHB –proteins/peptidesCHCA- lipids/peptidesDAN– lipidsApplication MethodSpraying (manual, robotic)SublimationSpotting (robotic)Data Processing
4IMS Performance Considerations Traditional MS Figures of MeritMass resolving power: m/ΔmMass accuracy: difference between measured mass and exact massSensitivity: overall response of the instrument for a given analyteDynamic range: range of detectable signalsMS/MS capabilities: ability to perform fragmentation experimentsSpecial IMS ConsiderationsSpatial resolution: distance between two neighboring pixels on the sample surfaceThroughput (acquisition rate): number of spectra acquired per unit timeData/file sizes: storage costs and processing abilities
5MALDI Imaging Platforms High mass resolution (m/Δm = 20,000)High repetition rate laser (5 kHz Nd:YLF)High digitizer acquisition rate ( pixels/sec)Continuous laser raster samplingMS/MSNext Generation MALDI TOFSimulTOF 200 Combo & 300 TandemMALDI TOFBruker Daltonics Autoflex IILITThermo LTQ XLFT-ICRBruker 9.4 T FTMSIM Q-TOFWaters Synapt G-2𝐿𝑎𝑡𝑒𝑟𝑎𝑙 𝑆𝑝𝑎𝑡𝑖𝑎𝑙 𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛=𝐻.𝐴 𝑣 𝑠𝑡𝑎𝑔𝑒 𝑓 𝑟𝑒𝑝J.M Spraggins and R.M. Caprioli, J. Am. Soc. Mass Spectrom. 2011, 22,
6Performance Tradeoffs Low Spatial Resolution (>100 μm)High Spatial Resolution (<10 μm)MinutesAcquisition TimeHours1 x 108Signal Intensity1 x 105<5 GBFile Size> 10GB
7#Pixels = f(Spatial Resolution, Area) High-spatial resolution and/or large area IMS experiments can require prohibitive analysis times.25 pixels2,500 pixels
8Pixel Size: The Importance of Speed ~21,000 pixels/3hr MALDI TOF~9,800 pixels/3hr LIT~7,700 pixels/3hr FT-ICR~7,200 pixels/3hr IM Q-TOF
12The Need for High Mass Resolution Average spectrum across tissue± 0.25 m/z1 mmSample: +Rat Brain (lipids)Laser Rep. Rate: 5000 HzLateral Spatial Resolution: 25 µmAcquisition Rate: 100 pixels/secondVertical Step: 25 µmPixels: 178,154 pixelsMatrix: CHCAAnalysis Time: ~90 minutes
13The Need for High Mass Resolution ± 0.25 m/zSample: +Rat Brain (lipids)Laser Rep. Rate: 5000 HzLateral Spatial Resolution: 25 µmAcquisition Rate: 100 pixels/secondVertical Step: 25 µmPixels: 178,154 pixelsMatrix: CHCAAnalysis Time: ~90 minutes~12,000 FWHM resolution at 869 m/z
14The Need for High Mass Resolution 869.32Δ = 0.19 m/z869.131 mm± 0.05 m/zcortex1 mm± 0.05 m/zcorpus callosumthalamushippocampus
15MALDI TOF/TOF Vg V Velocity and Space Focus Vp DetectorIon AcceleratorCollision CellTimed Ion SelectorHigh resolution timed ion selector (<5 Da)Collision cell for efficient fragmentationHigh repetition rate laser (5 kHz Nd:YLF)High digitizer acquisition rate ( pixels/sec)Continuous laser raster samplingMS/MS can provide a way to distinguish isobaric species and give sequence information on an analyte of interest.
19“Multiplexed” MS/MS Analysis Ion AcceleratorCollision CellTimed Ion SelectorHigh resolution timed ion selector (<5 Da)Selection of multiple precursor ions allows for multiple MS/MS transitions to be performed in a single laser shot.Monitoring MS/MS transitions is useful, but throughput is limited when only examining a single precursor ion.
22ConclusionsGoing from hours to minutes: High speed IMS produces high quality molecular images.Making the unseen, seen: Isobaric or near isobaric species require special consideration.High resolution MSMS/MSInforming biology: Coupled with the multiplexed nature of MS-based acquisition, the high throughput methodologies described herein offer viable means for studying complex biological systems in situ.
23Acknowledgements Mass Spectrometry Research Center SimulTOF Systems Richard Caprioli Kevin Schey Jeremy Norris Michelle Reyzer David Hachey Kristie Rose Andre Zavalin Jeff Spraggins Raf Van de Plas Erin Seeley Junhai Yang Peggi Angel Jere Compton Jamie Allen Brian Hachey Audra Judd Lisa Manier David Anderson Megan Gessel Dhananjay Sakrikar Domenico Taverna Kerri Grove Jessica Moore David Rizzo Chad Chumbley Jamie Wenke Faizan Zubair Kyle Floyd Monika MurphySimulTOF SystemsChristina Hsieh VestelGeorge MillsKenneth ParkerFundingNIH T32 ES007028NIH/NIGMS 5P41 GMMSACL Young Investigator Travel Award