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High Performance MALDI MS, MS/MS, and Multiplexed MS/MS Tissue Imaging. MSACL 2014 US March 1 - 5, 2014, San Diego, CA. Boone M. Prentice 1 , Kevin M. Hayden 2 , Marvin L. Vestal 2 , Richard M. Caprioli 1

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High performance maldi ms ms ms and multiplexed ms ms tissue imaging

High Performance MALDI MS, MS/MS, and Multiplexed MS/MS Tissue Imaging

MSACL 2014 US

March 1 - 5, 2014, San Diego, CA

Boone M. Prentice1, Kevin M. Hayden2, Marvin L. Vestal2, Richard M. Caprioli1

1Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, TN 37235

2SimulTOF Systems, Sudbury, MA 01776


Why imaging mass spectrometry
Why Imaging Mass Spectrometry? Tissue Imaging

IMS combines molecular specificity with location

cortex

Rat Kidney

medulla

783 m/z

773 m/z

704 m/z

726 m/z

733 m/z

742 m/z

761 m/z

821m/z

811m/z

805 m/z

797 m/z

787 m/z

809 m/z

849 m/z

The 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.


Imaging mass spectrometry
Imaging Mass Spectrometry Tissue Imaging

Matrix

application

Matrix-Assisted Laser Desorption/Ionization

Sample Preparation

Matrix

SA – proteins/peptides

DHB –proteins/peptides

CHCA- lipids/peptides

DAN– lipids

Application Method

Spraying (manual, robotic)

Sublimation

Spotting (robotic)

Laser

…………

…………

…………

…………

…………

…………

Lipid & Protein Ion Images

…………

…………

Data Processing

Images for individual m/z

values integrated over all pixels

MS spectrum for each x, y coordinate (pixel)


Ims performance considerations
IMS Performance Considerations Tissue Imaging

Traditional MS Figures of Merit

Mass resolving power: m/Δm

Mass accuracy: difference between measured mass and exact mass

Sensitivity: overall response of the instrument for a given analyte

Dynamic range: range of detectable signals

MS/MS capabilities: ability to perform fragmentation experiments

Special IMS Considerations

Spatial resolution: distance between two neighboring pixels on the sample surface

Throughput (acquisition rate): number of spectra acquired per unit time

Data/file sizes: storage costs and processing abilities


Maldi imaging platforms
MALDI Imaging Platforms Tissue Imaging

  • High mass resolution (m/Δm = 20,000)

  • High repetition rate laser (5 kHz Nd:YLF)

  • High digitizer acquisition rate (50-100 pixels/sec)

  • Continuous laser raster sampling

  • MS/MS

Next Generation MALDI TOF

SimulTOF 200 Combo & 300 Tandem

MALDI TOF

BrukerDaltonicsAutoflex II

LIT

Thermo LTQ XL

FT-ICR

Bruker 9.4 T FTMS

IM Q-TOF

Waters Synapt G-2

J.M Spraggins and R.M. Caprioli, J. Am. Soc. Mass Spectrom.2011, 22, 1022-1031.


Performance tradeoffs
Performance Tradeoffs Tissue Imaging

Low Spatial Resolution (>100 μm)

High Spatial Resolution (<10 μm)

Minutes

Hours

Acquisition Time

1 x 108

Signal Intensity

1 x 105

File Size

<5 GB

> 10GB


Pixels f spatial resolution area
#Pixels = f(Spatial Resolution, Area) Tissue Imaging

High-spatial resolution and/or large area IMS experiments can require prohibitive analysis times.

25 pixels

2,500 pixels


Pixel size the importance of speed
Pixel Size: The Importance of Speed Tissue Imaging

~21,000 pixels/3hr MALDI TOF

~9,800 pixels/3hr LIT

~7,700 pixels/3hr FT-ICR

~7,200 pixels/3hr IM Q-TOF


High speed maldi tof
High Speed MALDI TOF Tissue Imaging

~345,000 pixels/3hr Next Generation MALDI TOF

~21,000 pixels/3hr MALDI TOF

~9,800 pixels/3hr LIT

~7,700 pixels/3hr FT-ICR

~7,200 pixels/3hr IM Q-TOF


Ms protein imaging
MS Protein Imaging Tissue Imaging

Sample: +Mouse Kidney (infected w/ S. aureus)

Lateral Spatial Resolution: 25 µm

Vertical Step: 50 µm

Wash, Matrix:Carnoy’s, CHCA/DHB mix

Laser Rep. Rate: 1000 Hz

Acquisition Rate: 20 pixels/second

Pixels: 15,940 pixels

Analysis Time: ~20 minutes

~10,160 m/z

~15,000 m/z

1 mm

Red: S100A8 protein

Green:Hemoglobin alpha chain

Abscesses


Ms lipid imaging
MS Lipid Imaging Tissue Imaging

Average spectrum across tissue

Sample: +Rat Brain

Lateral Spatial Resolution: 50 µm

Vertical Step: 50 µm

Matrix: DAN

Laser Rep. Rate: 1000 Hz

Acquisition Rate: 20 pixels/second

Pixels: 39,073 pixels

Analysis Time: ~40 minutes

corpus callosum

fornix

striatum

ventricle

735 m/z

778 m/z

anterior commissure

1 mm

1 mm


The need for high mass resolution
The Need for High Mass Resolution Tissue Imaging

Average spectrum across tissue

1 mm

869.25 ± 0.25 m/z

Sample: +Rat Brain (lipids)

Lateral Spatial Resolution: 25 µm

Vertical Step: 25 µm

Matrix: CHCA

Laser Rep. Rate: 5000 Hz

Acquisition Rate: 100 pixels/second

Pixels: 178,154 pixels

Analysis Time: ~90 minutes


The need for high mass resolution1
The Need for High Mass Resolution Tissue Imaging

869.25 ± 0.25 m/z

Sample: +Rat Brain (lipids)

Lateral Spatial Resolution: 25 µm

Vertical Step: 25 µm

Matrix: CHCA

Laser Rep. Rate: 5000 Hz

Acquisition Rate: 100 pixels/second

Pixels: 178,154 pixels

Analysis Time: ~90 minutes

~12,000 FWHM resolution at 869 m/z


The need for high mass resolution2
The Need for High Mass Resolution Tissue Imaging

869.13

869.32

Δ = 0.19 m/z

cortex

thalamus

corpus callosum

hippocampus

1 mm

869.32 ± 0.05 m/z

1 mm

869.13 ± 0.05 m/z


Maldi tof tof
MALDI TOF/TOF Tissue Imaging

V

Velocity and Space Focus

Vg

Collision Cell

Ion Accelerator

Vp

Timed Ion Selector

Detector

  • High resolution timed ion selector (<5 Da)

  • Collision cell for efficient fragmentation

  • High repetition rate laser (5 kHz Nd:YLF)

  • High digitizer acquisition rate (50-100 pixels/sec)

  • Continuous laser raster sampling

MS/MS can provide a way to distinguish isobaric species and give sequence information on an analyte of interest.


Ms lipid imaging1
MS Lipid Imaging Tissue Imaging

869.2 ± 0.4 m/z

1 mm

Sample: +Rat Brain (lipids)

Lateral Spatial Resolution: 50 µm

Vertical Step: 50 µm

Matrix: DAN

Laser Rep. Rate: 1000 Hz

Acquisition Rate: 20 pixels/second

Pixels: 51,120 pixels

Analysis Time: ~45 minutes


Ms ms lipid imaging
MS/MS Lipid Imaging Tissue Imaging

MS/MS Precursor:869.2

Sample: +Rat Brain (lipids)

Lateral Spatial Resolution: 50 µm

Vertical Step: 50 µm

Matrix: DAN

Laser Rep. Rate: 1000 Hz

Acquisition Rate: 20 pixels/second

Pixels: 48,441 pixels

Analysis Time: ~45 minutes

hippocampus

1 mm

869 → 39 m/z

869 → 223 m/z

1 mm


Ms ms drug imaging
MS/MS Drug Imaging Tissue Imaging

Sample: +Rat Liver (synthetically does with Rifampicin, 400 mg liver immersed in 200 µM RIF solution for 100 hrs)

Lateral Spatial Resolution: 50 µm

Vertical Step: 50 µm

Matrix: THAP

Laser Rep. Rate: 1000 Hz

Acquisition Rate: 20 pixels/sec

Pixels: 14,192 pixels

Analysis Time: ~15 minutes

100%

0%

CID

[M-H]-

[M-H-424]-

821 m/z

397 m/z

1 mm

821 → 397 m/z


Multiplexed ms ms analysis
“Multiplexed” MS/MS Analysis Tissue Imaging

Collision Cell

Ion Accelerator

Timed Ion Selector

  • High 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.


Multiplexing
“Multiplexing” Tissue Imaging

Sample: +Rat Brain (lipids)

Lateral Spatial Resolution: 50 µm

Vertical Step: 50 µm

Matrix: DAN

Laser Rep. Rate: 1000 Hz

Acquisition Rate: 20 pixels/second

Pixels: 47,204 pixels

Analysis Time: ~40 minutes

827m/z

735 m/z

MS/MS of 735 m/z AND 827 m/z

MS Image

corpus callosum

hippocampus

827m/z

735 m/z

thalamus

735 m/z, 827 m/z

cortex

1 mm


Multiplexed imaging
Multiplexed Imaging Tissue Imaging

MS/MS of 735 m/z AND 827 m/z

827m/z

735 m/z

hippocampus

MS/MS Images

827m/z

735 m/z

corpus callosum

735 → 184 m/z

827 → 767 m/z

1 mm

1 mm


Conclusions
Conclusions Tissue Imaging

  • Going 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 MS

    • MS/MS

  • Informing 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.


Acknowledgements
Acknowledgements Tissue Imaging

Mass Spectrometry Research Center

SimulTOF Systems

Christina Hsieh Vestel

George Mills

Kenneth Parker

Funding

NIH T32 ES007028

NIH/NIGMS 5P41 GM103391-03

MSACL Young Investigator Travel Award

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 Murphy


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