Richard M. Caprioli
Mass Spectrometry Research Center Vanderbilt University
Imaging Mass Spectrometry:
Molecular Mapping Beyond the Microscope
NOTE: Confidential information for private viewing only
Mass spectrum from each pixel
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Laser ablation of array of x,y coordinate positions (pixels)
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Basic Approach of MALDI Imaging MS for Tissue Analysis
Mass Spectrometry Technology
1. Imaging/Profiling: MALDI TOF, TOF/TOF, LTQ, IM QTOF, FTICR ( Bruker Autoflex, Ultraflex II, 9.4T FTICR; Thermo LTQ; Waters Synapt; AB STR )
Laser Detector
Sample Plate
MALDI -TOF MS (linear)
2. Molecular ID: LC/MS/MS; LC or 2-D gel, MALDI MS/MS, IM QTOF (Bruker HCT, UltraflexII; Thermo LTQ; AB 4700; Waters Synapt )
Spatial Resolution 1 µm
Sensitivity (signal intensity, # peaks)
200 µm
Data File Size
Time of Acquisition
Imaging Mass Spectrometry - Tradeoffs Exptl Needs
Mouse Kidney – MALDI Imaging MS at 100 µm spatial resolution, 1 kHz rep rate laser, SA matrix
5 mm
(a) (b)
Human breast tumor cell line implanted into the tibia of a mouse. Human calcyclin (m/z 10,090) Mouse calcyclin (m/z 9960)
Erin Seeley, Lynn Matrisian
1342 1348 1354 1360 0
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IMS IHC
Substance P is predominantly localized to the ventromedial SNr
Malin Andersson, Ariel Deutch
300 504 708 912 1116 1320 Mass (m/z)
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Biological / Medical Research
Effects of Diabetes on the Kidney The Glomerulus
1,000,000 PER KIDNEY
Diabetic Nephropathy Goodpasture’s Disease Alport Syndrome
Kerry Grove, Megan Gessel, Billy Hudson
Human Kidney Glomerulus
10-20 years
NORMAL DIABETIC
DIABETICS High blood sugar
Kerri Grove, Billy Hudson
DAMAGE BY OXIDATIVE PATHWAYS INDUCED BY HYPERGLYCEMIA
High glucose
Glycated protein
Reactivecarbonyl species
Reactive oxygen species
Hydroxyl radical (●OH)
Hypohalous acids (HOCl , HOBr)
Glyoxal (CHO – CHO)
Methylglyoxal (CH3 –CO–CHO)
AGEs (advanced glycation end products)
AOEs
O2 / Mn+
High glucose
Glycated protein
(ΔM = +162)
Reactive carbonyl species
Reactive oxygen species
Hydroxyl radical (●OH)
Hypohalous acids (HOCl , HOBr)
Glyoxal (CHO – CHO)
Methylglyoxal (CH3 –CO–CHO)
Oxidation of Trp, Met, and Cys side chains
(ΔM = +16)
Chlorination and bromination of protein
amino groups (ΔM = +34, +79)
Carboxyethyllysine
(ΔM = +72)
Carboxymethyllysine (ΔM = +58)
5-Hydro- imidazolone (ΔM = +40)
Carboxymethyllysine (ΔM = +58)
Glucosepane (ΔM = +108)
O2 / Mn+
5-Hydro-5-methyl imidazolone (ΔM = +54)
Tetrahydropyrimidine (ΔM = +144)
PAS
MALDI IMS
m/z 4343
m/z 4415
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MS Images of Kidney in Wild Type and Diabetic Mouse
Green: wild type mouse Red : eNOS -/- db/db diabetic mouse
Fig. 2
FTICR MS - CASI (continuous accumulation of selected ions) to enhance dynamic range
Jeff Spraggins
HDR-MALDI FT-ICR mass spectrometry MALDI FT-ICR mass spectrometry
High Dynamic Range Analysis of a Kidney Tissue Section
HDR MALDI FT-ICR MS • Quad mass: m/z 900 • Isolation window: 20 Da • Laser Shots: 6000 • MS/MS used for IDs
Jeff Spraggins, Kerri Grove
Drug Penetration Into Tissue
Mass spectrum from lung tissue biopsy obtained on a low res MS (LTQ)
Study of Rifampicin Treated Rabbit Infected with TB
H&E stain Matrix
m/z 821.169
Rifampicin m/z 821.401
Accuracy: 3 ppm
Bacterial Lipid* PI(33:1)
m/z 821.521 Accuracy: 2.7 ppm
Lipid PG(40:6)
m/z 821.536 Accuracy: 2.2 ppm
*predicted Mycobacterium tuberculosis lipid from the species-specific LipidMaps Database.
Localization of Rifampicin in granulomas in TB infected rabbit lung 30 mg/kg oral dose daily for 1 week. MS/MS RIF [M-H]-
m/z 322 → 397 + 722
Optical image with granulomas indicated H&E image
MS/MS image of RIF (green) overlaid on optical image
1 mm
Comparison of MALDI and LC-MS
Lisa Manier, Clifton E. Barry, III
Drug Quantitation in Tissue - Mimetic Tissue Model
Tissue homogenates spiked with
range of drug concentrations Frozen polymer mold Cryosection and mount
adjacent to dosed tissue
Frozen Dosed
Tissue Slide for MALDI IMS
Courtesy Reid Groseclose and Steve Castellino, GSK
Drug Quantitation in Tissue
Courtesy Groseclose and Castellino, GSK
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SCH 412348 in rat brain: LC/MS vs. MALDI MS
Amount (ng/ml)
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MALDI (R2 = 0.93)
LC/MS (R2 = 0.95)
Michelle Reyzer, Walter Korfmacher (Schering-Plough)
Clinical Applications
Reid Groseclose
Serial Tissues Sections Mounted onto MALDI targets
Antigen Retrieval
In situ Trypsin Digestion
MALDI Imaging /Profiling Mass Spectrometry
Working with Formalin Fixed Paraffin Embedded Tissue
MALDI MS Profiling (Histology Directed Molecular Analysis)
Mass spectra mapped to specific locations in tissue based on histology image
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-- Fresh frozen tissue -- Formalin fixed paraffin embedded (in situ tryptic digestion)
Erin Seeley, Rossitza Lazova (Yale, Alirezer Zephir (Harvard))
MS Analysis of Spitzoid Lesions in FFPE Biopsies
Spitzoid Melanoma
Spitz Nevi
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Training set
# Patients
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Spitz nevi (SN) 26 100
Spitzoid Malignant Melanoma (SMM)
25 96
Classificat ion of Spitzoid Lesions
Validation (test) set
# Patients
Classification Accuracy (%)
Spitz nevi (SN) 30 97
Spitzoid Malignant Melanoma (SMM)
29 90
56 SN and 54 SMM from Yale University Spitzoid Neoplasm Repository
Primary Ocular Melanoma
Lesion on neck
Lesion on right arm
Case Study I
17 year old boy with ocular melanoma A few months later presented with two skin lesion Skin lesions clinically diagnosed as metastases
Tissue
Pathology
MS Analyses
(# separate areas) Biopsy Malignant
Melanoma
15/15 Malignant Melanoma
Mass Spectrometry Analysis
Tissue
Clinical
Evaluation
MS Analyses
(# separate areas)
Skin lesion #1 Malignant Melanoma
16/17Spitz nevus
Skin lesion #2 Malignant Melanoma
10/10Spitz nevus
Occular melanoma
Skin lesions
36 year old pregnant woman presents with lesion on upper arm
Excisional biopsy performed and determined to be malignant
Insufficient margins taken for size of lesion
No further treatment during pregnancy
Two months later, the baby was born with multiple nevi
Mother Mass Spectrometry
Malignant Melanoma 29/29 Malignant Melanoma
Skin lesions Baby
Mass Spectrometry
A indeterminate 23/23 Nevus
B indeterminate 9/9 Nevus
# Age Gender Site Histologic Dx MS dx Follow up (y) Clinical Status
1 43 M Back SMM SMM 3.5 Negative LN; ANED
2 23 F L calf SMM SMM 2 Positive LN; ANED
3 28 F Thigh SMM SMM 12 Positive LN 8 years later; ANED
4 6 F L neck SMM SMM 1.5 Positive LN; ANED
5 39 F L post leg SMM SMM 1.5 Positive LN; ANED
6 5 F Buttock SMM SMM 6 Positive LN; ANED
7 29 F R upper back SMM SMM 14 Negative LN: Re-excision; ANED;
8 50 M thorax SMM SMM 3 DOD with lung mets 3 years later
9 43 M back SMM SMM 4 Negative LN; ANED
10 57 F NK SMM SMM 3 Negative LN; ANED
LN – Lymph Node; ANED – Alive, No Evidence of Disease; DOD – Dead of Disease
11 15 F L neck SMM SN 4 Negative LN; ANED
12 6 M Abdomen SMM SN 1 ANED
13 44 F R upper arm SMM SN 7 ANED; 2 other ASN favor SN
14 16 M Back SMM SN 10 Negative LN; ANED
15 55 M R mid back SMM SN 2 ANED
16 40 F R upper arm SMM SN 11 Negative LN; ANED
17 9 M R upper arm SMM SN 14 Negative LN; ANED
18 17 M Chest SMM SN 1 Negative LN; ANED
19 54 F R upper arm SMM SN 8 Negative LN; ANED
20 44 F
R buttock SMM SN 9 Negative LN; ANED
R upper arm SMM SN 8 Negative LN; ANED
21 30 F R shin SMM SN 14 ANED
22 57 M R thigh SMM SN 12 ANED
23 46 M R arm SMM SN 4 1 Positive LN-1 cell; ANED
24 54 F R upper arm SMM SN 8 Negative LN; ANED
# Age Gender Site Histologic Dx MS dx Follow up (y) Clinical Status
1 43 M Back SMM SMM 3.5 Negative LN; ANED
2 23 F L calf SMM SMM 2 Positive LN; ANED
3 28 F Thigh SMM SMM 12 Positive LN 8 years later; ANED
4 6 F L neck SMM SMM 1.5 Positive LN; ANED
5 39 F L post leg SMM SMM 1.5 Positive LN; ANED
6 5 F Buttock SMM SMM 6 Positive LN; ANED
7 29 F R upper back SMM SMM 14 Negative LN: Re-excision; ANED;
8 50 M thorax SMM SMM 3 DOD with lung mets 3 years later
9 43 M back SMM SMM 4 Negative LN; ANED
10 57 F NK SMM SMM 3 Negative LN; ANED
11 15 F L neck SMM SN 4 Negative LN; ANED
12 6 M Abdomen SMM SN 1 ANED
13 44 F R upper arm SMM SN 7 ANED; 2 other ASN favor SN
14 16 M Back SMM SN 10 Negative LN; ANED
15 55 M R mid back SMM SN 2 ANED
16 40 F R upper arm SMM SN 11 Negative LN; ANED
17 9 M R upper arm SMM SN 14 Negative LN; ANED
18 17 M Chest SMM SN 1 Negative LN; ANED
19 54 F R upper arm SMM SN 8 Negative LN; ANED
20 44 F
R buttock SMM SN 9 Negative LN; ANED
R upper arm SMM SN 8 Negative LN; ANED
21 30 F R shin SMM SN 14 ANED
22 57 M R thigh SMM SN 12 ANED
23 46 M R arm SMM SN 4 1 Positive LN-1 cell; ANED
24 54 F R upper arm SMM SN 8 Negative LN; ANED
LN – Lymph Node; ANED – Alive, No Evidence of Disease; DOD – Dead of Disease
# Age Gender Site Histologic Dx MS dx Follow up (y) Clinical Status 1 35 F L thigh ASN SN 5.5 ANED
2 33 M Back ASN SN 7 ANED
3 14 M L arm ASN SN 7 ANED
4 11 M Posterior neck ASN SN 7 ANED
5 55 F L upper back ASN SN 7 ANED
6 3 M R ear ASN SN 7 ANED
7 16 M R canthus ASN SN 6 ANED
8 34 M R leg ASN SN 1 ANED
9 7 M Scalp ASN SN 5 ANED
10 28 F Breast ASN SN 6 ANED
11 18 M Neck ASN SN 2.5 0/1 SLN; ANED
12 39 F R ankle/foot ASN SN 6 ANED
13 34 F R shin ASN SN 6 ANED
14 72 F L arm ASN SN 6 ANED
15 13 F R medial knee ASN SN 6 ANED
16 22 F L ant thigh ASN SN 6 ANED
17 49 F L medial thigh ASN SN 5 0/1 SLN; 0/1 LN; ANED
18 30 F R thigh
ASN - orig. bx SN
5 ANED ASN - re-exc. SN
19 3 M R cheek ASN SN 5 ANED
20 26 F R thigh ASN SN 5 ANED
21 38 M R cheek ASN SN 5 ANED
22 6 F R medial thigh ASN SN 5 ANED
23 8 M R extensor elbow ASN SN 5 ANED
24 19 F R upper thigh ASN SN 1 ANED
25 45 M R calf ASN SN 1 0/3 SLN; 0/1 LN; ANED
26 58 F L upper back ASN SN 1 ANED
27 29 F L buttock ASN SN 1 ANED
28 9 M R lateral knee ASN SN 6 1+SLN; 0/1 LN; CGH-no abnormalities; ANED
29 16 M L ear ASN SN 8 ANED
30 4 F R thigh ASN SN 8 ANED
31 27 F L shin ASN SN 15 ANED
32 34 M NK ASN SN 14 ANED
33 48 M L flank ASN SN 13 ANED
34 34 F R post forearm ASN SN 11 ANED
35 37 F R ear ASN SN 6 ANED
36 7 F R lower back ASN SN 15 ANED
37 24 F L buttock ASN SN 14 ANED
38 39 M R post arm ASN SN 6 ANED
New Technology Initiatives
High Spatial Resolution Imaging (1-5 μm) Increased Imaging Speed (<< 1 sec/pixel) Multi-Modal Image Fusion 3-D MS Images Increased Sensitivity – Targeted Analyses, Derivatization Ease of Use: Matrix Pre-Coated Targets
MALDI (TOF) IMS using a 5 kHz Nd: YAG continuous laser
m/z 734.4 ( PC 32:0)
m/z 788.5 (PC 36:1)
m/z 806.5 (PC 38:6)
lipid images acquired at a rate of 30 pixels/s Total image time: < 8 min
Jeff Spraggins
High Spatial Resolution Imaging MS
(1 - 5 µm pixel diameter)
Schematic of a Transmission Geometry Instrument
Andrey Zavalin Ken Shriver
Single Cell / Sub-cellular Imaging Pancreas islet section ablated with 30 shots from a 2-3 µm diameter laser spot on target
Andrey Zavalin, Kerri Grove
5400 6400 m/z
m/z 750 PE (18:0p/20:4) m/z 1052 Hex-Sulfo-Hex-Cer-(d16:1/26:0) m/z 906 SulfoHex-Cer (t18:0/24:1)
100 mm
Ion Images of Human Kidney Cortex Resolution: 1 mm laser beam, 2 mm pitch
25 shots/pixel, transmission geometry matrix sublimed DAN
Analysis of Single Mammalian Cells
HEK-293 cells
MS image: m/z 782
Optical image After ablation
RKO cells
MS image: m/z 782 Optical image after ablation
Image resolution: 1 µm beam diameter at 1.5 µm pitch; 349 nm UV laser; rep rate 1 kHz
Erik Todd, Andrey Zavalin
1pixel 25 shots
25 shots
Single Cell Analysis – HEK 293
Transmission geometry TOF MS, Nd:YLF laser (355 nm), single shot
1 µm laser beam
5 µm laser beam
10 µm 10 µm
Image Processing
IMAGE FUSION
TISSUE
imaging MS
microscopy
tagged
microscopy
NMR
CT
PET
Low spatial res.
High chemical info
High spatial res.
Low chemical info
…combines best attributes of imaging technologies …can highlight non-obvious relationships with other technologies (e.g., MRI, PET, microscopy, etc.) …is predictive in nature
A page from satellite imaging...
pan-chromatic image - high spatial resolution - little info per pixel
multispectral image - low spatial resolution - a lot of info per pixel
fused image - high spatial resolution - a lot of info per pixel
IMS – Microscopy Fusion
Creating Multi-Modal Images
Raf Van de Plas
Imaged at 100 um
Microscopy 5 µm MS Image at 100 µm
IMAGE FUSION Mass Spectrometry and Microscopy
Raf Van de Plas
Predictive MS image sharpening through fusion with microscopy m/z 18,399 - transverse section of mouse brain
MS Fused Image sharpened to 5 µm
m/z 18,399
3–D Imaging Mass Spectrometry
3D Imaging MS: Correlation with MRI
Erin Seeley Tuhin Sinah John Gore
H&E staining of the mouse pup - progression from superficial sections containing just skin, muscle, and bone through the abdominal organs and central nervous system and back to superficial sections.
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Preparation for 3D Image of Whole Mouse Pup 15 µm sections at 210 µm spacing
Erin Seeley, Tuhin Sinha
Erin Seeley, Eric Skaar
Systemic Staph Infection in Mouse
Erin Seeley, Kaitlin Schroeder, Eric Skaar, Kevin Wilson
Systemic Staph Infection in Mouse Kidney m/z 10,165 (Calgranulin A) located in abscesses
Perspectives
Instrumental / Methodology Challenges
Sensitivity - achieve more global coverage ( fraction of proteome now observed) Resolution - better lateral resolution (routine single cell imaging) - higher MS resolution (better resolve isoforms, PTMs, isobars) Mass Range - routinely beyond 50 kd Identification - in situ - fast, simple, accurate Quantitation - reagents and methods - isotope based, relative and absolute Validation - cross-lab (std protocols), cross-platform reproducibility/standardization Availability - single manufacturer must provide entire technology ‘solution’
IMS - Summary
- measures native molecular distributions, providing new biological insights that easily correlate with other imaging modalities - is an excellent discovery technology because no target specific reagents are needed - has exceptionally high throughput (in some cases less than a few seconds for data acquisition per sample), providing multiple images simultaneously at discrete MW values - can undergo fusion with data from other imaging modalities to create new imaging paradigms
Mass Spectrometry Research Center Jeremy Norris Erin Seeley Michelle Reyzer Andrey Zavalin Jeff Spraggins Peggi Angel Lisa Manier Junhai Yang Kerri Grove Raf Van de Plas Megan Gessel David Anderson Brian Hachey Boone Perkins
Vanderbilt Collaborators David Hachey Kevin Schey Paul Laibinis John Gore Charles Manning Eric Skaar Billy Hudson Nancy Brown Randy Blakely Anna Carneiro Ariel Deutch Ray Mernaugh Melinda Sanders Kay Washington Kevin Wilson
Funding NIH GMS – 3D Imaging NCRR/GMS - National Resource for IMS Department of Defense The Gates Foundation Vanderbilt University
Others Peter Wild, U Zurich Reid Groseclose, GlazoSmithKline Kristin Burnum, Batelle PNW Labs John Mayer, Harvard Pierre Chaurand, U Montreal Shannon Cornett, Bruker Daltonics Ron Kahn, Harvard Andre Kleinridders, Harvard SK Dey, U Cincinnati Giovanni Sindona, U Calabria Alireza Sepehr (Harvard) Rossitza Lazova (Yale) Gwendoline Thiery, Harvard Kristina Schwamborn, Univ. Munich
Thank You
