Tissue Imaging by 5 kHz High-Performance MALDI-TOF Poster Number TP191 Christina Vestal 1, Kenneth...
1
Tissue Imaging by 5 kHz High-Performance MALDI-TOF Poster Number TP191 Christina Vestal 1 , Kenneth Parker 1 , Kevin Hayden 1 , George Mills 1 , Marvin Vestal 1 , Shannon Cornett 2 , and Richard Caprioli 2 . 1 Virgin Instruments, Sudbury, MA, 2 Vanderbilt University School of Medicine, Nashville, TN. Introduction Tissue imaging and profiling by MALDI-TOF MS provides unique information that facilitates our understanding of normal biological and pathological processes. This technology provides a high- throughput tool to interrogate protein expression in tissue at the molecular level. It can also provide direct measurements of distribution of drugs and metabolites in tissues and correlate these distributions with changes in the proteome. While many significant results have been reported over the past ten years, more widespread applications have been inhibited by limitations on the combined speed and spatial resolution available with existing instruments. In this work two new high-performance MALD-TOF instruments are evaluated for these applications. Method The new analyzers employ a 5 kHz laser for higher speed and better sample utilization. A linear MALDI-TOF mass spectrometers is used for imaging proteins. This analyzer operates at ion energy of 42 keV and includes a new hybrid detector that is immune to saturation effects without compromising time resolution. The second instrument is a reflector MS operating at 8 kV that provides very high resolving power and mass accuracy for peptides and small molecules. This instrument was used for imaging lipids in tissue samples using DHB matrix Sample Plates These instruments employ a sample plate 124x127 mm outside dimensions with 102x108 mm active area, allowing relative large samples such as whole body tissue sections from small mammals to be imaged with relatively high resolution. For this work adaptor plates accommodating up to 4 conductive 45x45 glass slides or 45x47 metal plates were used. An image of the plate is generated by a flat-bed scanner and that image is used to define the sample to be analyzed. Conclusions and Future Work This work has demonstrated that tissue images with 50 m resolution can be generated with practical speeds for many applications using a laser operating at 5 kHz. There is no apparent loss in data quality relative to earlier work at 20-200 Hz. We have demonstrated that up to 1 million spectra can be generated, stored, and processed per day, but at present data interpretation may be significantly slower than data acquisition. The images and spectra presented here represent a very small part of the total information available in one of these measurements. Further improvements in software and matrix deposition will be required to fully utilize this capability. References 1. J. A. Hankin, R. M. Barkley, R. C. Murphy, “Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging”, J. Am. Soc. Mass Spectrom. 2007, 18, 1646-1652. 2. P. Chaurand, J. L. Norris, D. S. Cornett, J. A. Mobley, and R. M. Caprioli, “New Developments in Profiling and Imaging of Proteins from Tissue Sections by MALDI Mass Spectrometry”, J. Proteome Res. 2006, 5, 2889-2900. Acknowledgements The assistance of Dr. Pierre Chaurand in providing the tissue sections with matrix deposition and the histological images of the sections. This work was supported in part by the National Institutes of Health under grants Area Chromatogram -A ll Runs -db:///Marvin/Vanderbilt/Lipids3 -52887.026 s.d. with 96 %hits 20 30 40 35 40 XP osition Y P osition Area Chromatogram -Unknown Traversal -db:///Marvin/shannon/lipid1La 50 MostIntense V?task=0 (S pectrumDataR educer)Reduced -14.658 s.d.w ith 56 %hits 75 80 85 90 84 85 86 87 88 89 90 91 X P osition YP osition 772.6 /(798 -802) 866.7 /(798 -802) 782.7 /(798 -802) A rea Chrom atog ram -A ll Runs -db:///M arv in/RichardV isit/linprot2 - 33.598 s.d . w ith 37 % hits 70 80 90 100 25 30 X P osition Y P osition 8563 /(3000 -30000) 12124 /(3000 -30000) 14044 /(3000 -30000) 10158 /(3000 -30000) 15012 /(3000 -30000) 10894 /(3000 -30000) 4964 /(3000 -30000) 12414 /(3000 -30000) 70 80 90 100 25 30 35 X P osition Y P osition 70 80 90 100 25 30 X P osition Y P osition U ntitled 7748 8377 15461 16202 24074 31829 47359 63028 78637 0.000 0.002 0.004 V olts G roup:4 Spot:13560 Shots:3000 6112 6593 7354 7932 8554 8978 9322 9973 11328 12123 14041 15004 15737 5968 10968 15968 0.000 0.002 0.004 0.006 0.008 D altons V olts G roup:4 Spot:15915 Shots:500 \\domainserver\public\Marvin\MiniTof-2009_03_18_13_51\MiniTof-0007-2009_03_18_13_53_53.330-.sqlspectrum#2951 798.55 799.56 800.56 760.58 826.57 772.54 848.63 782.57 828.54 806.52 830.52 850.62 734.57 822.57 852.58 866.65 762.60 783.56 768.55 790.56 756.54 834.58 735.57 804.49 767.56 78.004443 80.004443 82.004443 84.004443 86.004443 0.0 0.5 1.0 1.5 Microseconds V olts \M iniTof-2009_03_18_13_51\M iniTof-0007-2009_03_18_13_ 868.54 866.72 864.71 870.55 866.54 871.56 868.72 864.54 870.77 84.8000000 85.0000000 0.0 0.2 0.4 M icroseconds Volts A rea C hrom atogram - A ll R uns -db:///M arvin/R ichardV isit/linprot2 - 3130.757 s.d. with 58 % hits 70 80 90 100 25 30 35 X Position Y P osition 10000 -15020 A rea C hrom atogram - A ll R uns - db:///M arvin/RichardV isit/linprot2 - 51.142 s.d.w ith 4 % hits 70 80 90 100 25 30 35 X P osition Y P osition 16768 / (10000 -15020) 22159 / (10000 -15020) Untitled Linear-Linear 4748 4937 4964 5105 5155 7021 7508 7537 8562 8602 10157 10204 10270 10396 10463 10863 10938 10975 11324 11367 12960 13000 13063 14000 14042 14994 15120 15264 16767 5000 10000 15000 20000 0.000 0.002 0.004 0.006 0.008 D altons Volts Group:4 S pot:17036 S hots:3000 Untitled Linear-Linear 8978 9905 9972 10253 11324 11535 12362 14009 14046 14998 16773 16808 22159 10000 15000 20000 25000 0.0000 0.0005 0.0010 0.0015 D altons V olts G roup:4 Spot:15522 Shots:22000 Area C hrom atogram -A llR uns - db:///M arvin/RichardVisit/linprot2 -5915.735 s.d. with 80 % hits 95.0 95.5 96.0 96.5 27.2 27.4 27.6 27.8 X P osition Y P osition 3000 -30000 Area C hrom atogram -A ll R uns - db:///M arvin/R ichardV isit/linprot2 - 11507.209 s.d. with 76 % hits 70 80 90 100 25 30 X P osition Y P osition 2000 -30000 U ntitled Linear-Linear 2787 2825 2854 4111 4150 4180 4219 5351 5462 5607 6211 6248 6967 7260 8439 11202 11255 11320 11400 12384 12418 12488 12627 12689 5000 10000 0.00 0.01 0.02 D altons Volts G roup:4 Spot:13037 Shots:20500 TIC 500-1000 772.6 866.6 50 m resolution 1 2 3 4 protein The metal plate in the upper left position contains three mouse brain sections similar to those analyzed here, but all of the work presented was from samples on conductive glass plates. Whole mouse pup sections labeled 1-4 were analyzed for lipids using sublimed DHB matrix as described earlier. 1 This image was generated after analysis and most of the matrix has been desorbed. All 4 of these sections were analyzed in one day and generated 1,000,000 mass spectra. Data from one of these (3) is presented here. All of the data presented on protein imaging Example of mouse brain image generated with profile spectra. 250 shots per pixel, 1 mm/s raster at 50 m spacing. 100,000 spectra in 1.4 hours 20,000 RP note doublets High Speed Imaging of lipids in mouse brain sections. 2.5 mm/s, raster at 50 m spacing, 50 shots/pixel (spectra transfer time 0.01 s) 50 pixels/s at 50 m resolution 150,000 spectra in 50 minutes stained section for similar (but not identical) mouse brain section 772.6 772, 800,845,881 Lipid images of whole mouse pup section at 50 m resolution, 100 shots/pixel, 150,000 spectra in 1.3 hours. Note: Our mouse is backside down. stain Protein images of whole mouse pup at 5 kHz, 37 J/pulse. Raster at 50 m spacing, 1 mm/s 500 shots/spectrum, 50x100 m pixels, 75,000 spectra in 2.2 hours. Expanded view of 1x1.5 mm portion of TIC for whole mouse image. Sinipinic acid matrix was spotted at 200 m intervals and sample was scanned with 50x100 m pixels. Results indicate that matrix spots typically less than 150 m in dia and resolution of scanning is 50 m. 200 m brain bladder spine spinal cord Imaging of lipids and other small molecules using high-resolution Reflector TOF employs “on-the-fly” peak detection and calibration. Spectra are only saved if they meet predetermined criteria; typically a minimum intensity within a selected mass range. This has no effect on maximum acquisition rate (limited only by transfer time from digitizer to computer--typically <10 ms) but dramatically reduces storage requirements and accelerates data reduction and interpretation. Protein imaging with the linear TOF typically employs full profile spectra with 4 ns bins
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Transcript of Tissue Imaging by 5 kHz High-Performance MALDI-TOF Poster Number TP191 Christina Vestal 1, Kenneth...
Tissue Imaging by 5 kHz High-Performance MALDI-TOF Poster Number TP191
Christina Vestal1, Kenneth Parker1, Kevin Hayden1, George Mills1, Marvin Vestal1, Shannon Cornett2, and Richard Caprioli2. 1Virgin Instruments, Sudbury, MA, 2Vanderbilt University School of Medicine, Nashville, TN.
IntroductionTissue imaging and profiling by MALDI-TOF MS provides unique information that facilitates our understanding of normal biological and pathological processes. This technology provides a high-throughput tool to interrogate protein expression in tissue at the molecular level. It can also provide direct measurements of distribution of drugs and metabolites in tissues and correlate these distributions with changes in the proteome. While many significant results have been reported over the past ten years, more widespread applications have been inhibited by limitations on the combined speed and spatial resolution available with existing instruments. In this work two new high-performance MALD-TOF instruments are evaluated for these applications. MethodThe new analyzers employ a 5 kHz laser for higher speed and better sample utilization.
A linear MALDI-TOF mass spectrometers is used for imaging proteins. This analyzer operates at ion energy of 42 keV and includes a new hybrid detector that is immune to saturation effects without compromising time resolution.
The second instrument is a reflector MS operating at 8 kV that provides very high resolving power and mass accuracy for peptides and small molecules. This instrument was used for imaging lipids in tissue samples using DHB matrixSample PlatesThese instruments employ a sample plate 124x127 mm outside dimensions with 102x108 mm active area, allowing relative large samples such as whole body tissue sections from small mammals to be imaged with relatively high resolution. For this work adaptor plates accommodating up to 4 conductive 45x45 glass slides or 45x47 metal plates were used. An image of the plate is generated by a flat-bed scanner and that image is used to define the sample to be analyzed.
Conclusions and Future WorkThis work has demonstrated that tissue images with 50 m resolution can be generated with practical speeds for many applications using a laser operating at 5 kHz. There is no apparent loss in data quality relative to earlier work at 20-200 Hz. We have demonstrated that up to 1 million spectra can be generated, stored, and processed per day, but at present data interpretation may be significantly slower than data acquisition. The images and spectra presented here represent a very small part of the total information available in one of these measurements. Further improvements in software and matrix deposition will be required to fully utilize this
capability.References1. J. A. Hankin, R. M. Barkley, R. C. Murphy, “Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging”, J. Am. Soc. Mass Spectrom. 2007, 18, 1646-1652.2. P. Chaurand, J. L. Norris, D. S. Cornett, J. A. Mobley, and R. M. Caprioli, “New Developments in Profiling and Imaging of Proteins from Tissue Sections by MALDI Mass Spectrometry”, J. Proteome Res. 2006, 5, 2889-2900.AcknowledgementsThe assistance of Dr. Pierre Chaurand in providing the tissue sections with matrix deposition and the histological images of the sections.
This work was supported in part by the National Institutes of Health under grants RR025705 and GM079833.
Area Chromatogram - All Runs - db:///Marvin/Vanderbilt/Lipids3 - 52887.026 s.d. with 96 %hits
20 30 40
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osit
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Area Chromatogram - Unknown Traversal - db:///Marvin/shannon/lipid1La 50 Most Intense V?task=0 (SpectrumDataReducer) Reduced - 14.658 s.d. with 56 %hits
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Area Chromatogram - All Runs - db:///Marvin/RichardVisit/linprot2 - 33.598 s.d. with 37 %hits
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Area Chromatogram - All Runs - db:///Marvin/RichardVisit/linprot2 - 6982.828 s.d. with 42 %hits
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Area Chromatogram - All Runs - db:///Marvin/RichardVisit/linprot2 - 286.258 s.d. with 7 %hits
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Area Chromatogram - All Runs - db:///Marvin/RichardVisit/linprot2 - 3130.757 s.d. with 58 %hits
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Area Chromatogram - All Runs - db:///Marvin/RichardVisit/linprot2 - 51.142 s.d. with 4 %hits
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Area Chromatogram - All Runs - db:///Marvin/RichardVisit/linprot2 - 11507.209 s.d. with 76 %hits
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50 m resolution
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4protein
The metal plate in the upper left position contains three mouse brain sections similar to those analyzed here, but all of the workpresented was from samples on conductive glass plates. Wholemouse pup sections labeled 1-4 were analyzed for lipids usingsublimed DHB matrix as described earlier.1 This image was generated after analysis and most of the matrix has been desorbed. All 4 of these sections were analyzed in one day and generated 1,000,000 mass spectra. Data from one of these (3) is presented here.
All of the data presented on protein imaging from whole mouse pup was generated from the sample on a glass slide at lower right. Matrix was spotted with sinipinic acid at 200 mm intervals using the automatic spotter described previously.2
Example of mouse brainimage generated withprofile spectra. 250 shotsper pixel, 1 mm/s raster at50 m spacing. 100,000spectra in 1.4 hours
20,000 RPnote doublets
High Speed Imaging of lipids in mouse brain sections. 2.5 mm/s, raster at 50 m spacing, 50 shots/pixel (spectra transfer time 0.01 s) 50 pixels/s at 50 m resolution 150,000 spectra in 50 minutes
stained section for similar (but not identical)mouse brain section
772.6
772, 800,845,881
Lipid images of whole mouse pup section at 50 m resolution, 100 shots/pixel, 150,000 spectra in 1.3 hours. Note: Our mouse is backside down.
stain
Protein images of whole mousepup at 5 kHz, 37 J/pulse.Raster at 50 m spacing, 1 mm/s 500 shots/spectrum, 50x100 m pixels, 75,000 spectra in 2.2 hours.
Expanded view of 1x1.5 mmportion of TIC for whole mouse image. Sinipinic acid matrix was spottedat 200 m intervals and sample was scannedwith 50x100 m pixels. Results indicate thatmatrix spots typically less than 150 m in diaand resolution of scanning is 50 m.
200 m
brainbladder spine spinal cord
Imaging of lipids and other small molecules using high-resolution Reflector TOF employs“on-the-fly” peak detection and calibration. Spectra are only saved if they meet predetermined criteria; typically a minimum intensity within a selectedmass range. This has no effect on maximumacquisition rate (limited only by transfer timefrom digitizer to computer--typically <10 ms) butdramatically reduces storage requirements and accelerates data reduction and interpretation.Protein imaging with the linear TOF typically employs full profile spectra with 4 ns bins
