R. Graham Cooks, Cedric D’hue, Allison Dill, Demian Ifa,

ACCELERATING CLINICAL AND TRANSLATIONAL RESEARCH

www.indianactsi.org

R. Graham Cooks, Cedric D’hue, Allison Dill, Demian Ifa, Livia Eberlin, Christina Ferreira, Alan K. JarmuschKevin Kerian, Valentina Pirro

Department of Chemistry and Center for Analytical Instrumentation Development,Bindley Biosciences Center, Discovery Park, Purdue University

Hospital EngagementSurgery

Hospital Collaborators: Nathalie Agar, Brigham & Women’s HospitalTimothy Masterson, Michael Koch, Liang Cheng, IUPUI


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DESI Imaging

7 mm

“Cat on a pillow”

m/z 443

Resolution ~ 50 µm


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b) Line Scans

Signal ratios from MS Image construction

m/z 788

m/z 448

c) Tissue Imaging

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25

log (

abu

ndan

ce ra

tio o

f m/z

788

: 44

8)

Lateral distance (mm)

Abundance of Phosphatidylserine in Human Liver Adenocarcinoma

tumor

nontumor

Tumor margin

Justin M. Wiseman, Satu M. Puolitaival, Zoltán Takáts, R. Graham Cooks, Richard M. Caprioli, Angew. Chem. 2005

Tissue Analysis : Metabolite Profiling a) One Pixel


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Brain Tumor Diagnosis by Mass Spectrometry

L.S. Eberlin, et al. Proc. Natl. Acad. Sci. 110, 1611 (2013)

Differentiation of tumor subtype, grade, and tumor cell concentration

Endogenous lipid profiles


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Human Brain CancersHuman Brain Cancers

• Over 125 types of brain tumors – complex to diagnose• Mean survival 12 – 15 months to some years • New cases (US): 22,910/year, deaths: 13,700/year• Neurosurgery is the primary treatment • Critical decisions made during neurosurgery

Need: Improved intraoperative diagnosis of tumors & margins One Answer: Direct tissue analysis! – Molecular histology using Mass

Spec


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Human Brain Gliomas – Subtypes

Same W.H.O grade (III), different

subtypes

Gemistocytic

L.S. Eberlin, I. Norton, et al Cancer Research, 2012, 72, 645


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Human Brain Gliomas – Classification models• A model creation dataset for 3

classifiers was constructed (type, grade, %) using training set

Overall Recognition

CapabilityCross

ValidationSubtype 99.3 % 97.9 %Grade 99.7 % 99.2 %

Concentration 99.0 % 97.5 %

Independent Validation Set Class imaging Results (color coded)

Sample G26Grade IV

astrocytoma

Sample G33Grade IV

astrocytoma(infiltrative)

L.S. Eberlin, I. Norton, et al Cancer Research, 2012, 72, 645


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Prostate Disease State Touch Spray MS

-25 -20 -15 -10 -5 0 5 10 15

-15

-10

-5

0

5

10

15

20

359

10

11 12131416

17

1819

2123

29

30 31

32

3335

363738

3941

424546

4748 49

5051

52

53

5455 57

58

59

6062

63

64656667

68

69

70

71

72

73

747780

81

82

83

84

8586

88

89 91

92

93

9495

96 9798 99

100 12

4

6

7

8

15

20

22

2425

26 2728

3440 434456

61

75

76

78

79

8790

Scores on PC3 (13.4%)

Score

s o

n P

C4 (

6.2

%)

SCORE PLOT

Kevin Kerian, Alan Jarmusch, Valentina Pirro -------Purdue Timothy Masterson, Michael Koch, Liang Cheng--- IUPUI

m/z 700 - 1000

3 years of data; dozens of patients

ONE PIXEL


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IUSM – DESI - Prostate

720 740 760 780 800 820 840 860 880

m/z

0

1000

100

Rel

ati

ve

Ab

un

da

nc

e

788.4

885.5

786.4

861.6812.4 835.3723.4 816.5810.3747.4

788.4

885.5

812.4834.4

723.5 786.5 810.4861.5747.6

Cancer

Normal

H&E

788.5

861.5

0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60Time (min)

0

100

Rel

ati

ve

Ab

un

da

nc

e 788.5861.5

PI(38:4)PS(36:1) PI(36:4)

PI(34:5)


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MS Pathology: Prostate

-25 -20 -15 -10 -5 0 5 10 15

-15

-10

-5

0

5

10

15

3

5

9

10 11

12

13

1416

17

18

19

21

23

2930

3132 33

3536 37

3839

4142

45

46

47

48

49

50

51525354

55

57 58 5960

62

63

64 6566

6768

69

70

717273

74

77

80

8182

83

84 8586

88

89

91

92

93

94

95

96

97

98

99

100

1

2

4

678

15 20

22

24

25

262728

34

4043

44

56

61

75

76

7879

87

90


Scor

es o

n PC

3 (6

.1%

)

SCORE PLOT

(AVG) CV prediction rate = 95%

-25 -20 -15 -10 -5 0 5 10 15

-15

-10

-5

0

5

10

15

20

359

10

11 12131416

17

1819

2123

29

30 31

32

3335

363738

3941

424546

4748 49

5051

52

53

5455 57

58

59

6062

63

64656667

68

69

70

71

72

73

747780

81

82

83

84

8586

88

89 91

92

93

9495

96 9798 99

100 12

4

6

7

8

15

20

22

2425

26 2728

3440 434456

61

75

76

78

79

8790


Scor

es o

n PC

4 (6

.2%

)

SCORE PLOT

(AVG) CV prediction rate = 96%

DESITouch Spray

Diagnosis:Tumor or Normal


DESI-MS ImagingDESI-MS Imaging

Lack of Analytical (Molecular)

Diagnosis Methods


Diagnosis MethodsPro

blem

Pro

blem

Too l

Too l

Goa

lG

oal

TS-MSTS-MS

Too l

Too l

Alan Jarmusch, Kevin Kerian, Valentina Pirro12 patients : 100 samples+

6


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TS_RCC_3142014_Point1 #6-9 RT: 0.06-0.08 AV: 4 NL: 2.65E4T: ITMS - p NSI Full ms [150.00-2000.00]

200 400 600 800 1000 1200m/z

0

10

20

30

40

50

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70

80

90

100

Rela

tive

Abun

danc

e

247.0

187.1

788.2

898.3737.3 950.1736.2

626.1 951.1595.5 1060.2593.5 700.5279.3 583.3 1062.1996.8

MS Pathology: Kidney



DESI-MS ImagingDESI-MS Imaging


Diagnosis Methods


Diagnosis MethodsPro

blem

Pro

blem

Too l

Too l

Goa

lG

oal

TS-MSTS-MS

Too l

Too l

1 patient

Kevin Kerian, Valentina Pirro

RT: 0.00 - 0.69

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Time (min)

0

5

10

15

20

25

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45

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60

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Rela

tive

Abun

danc

e

0.64

0.28

0.10

0.570.07 0.480.12 0.56

0.25 0.44

NL:1.58E7

TIC MS TS_RCC_3142014_Point1

6 seconds

TSCase 1

*actual photo

TS chronogramTS spectrum (avg. 3 scans)

* many peaks – assumed to be adducts


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MS Pathology: KidneyKevin Kerian,

Valentina PirroAlan Jarmusch

TS Case 1 – directly from surgically resected tissue


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Blastocysts developing

In vitro

DESI-MS Signatures in Bovine Early Embryonic DevelopmentDESI-MS Signatures in Bovine Early Embryonic Development

Negative ion modeNegative ion mode

Immature oocytes

Eggs matured in vitro

Positive ion mode (silver adducts)Positive ion mode (silver adducts)

Blastocysts Developing In vivo

Fertilization

Christina Ferreira,PLoS One. 2013 20;8(9):e74981


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Ambient Ionization for Early Detection of Parkinson’s Disease in Cerebrospinal fluid

Compound Concentration RSD

Methionine5 ppm in H2O 11%

10 ppm in H2O 17%

Arginine5 ppm in H2O 3%

10 ppm in H2O 3%Karen Cesafsky and Ryan Espy

Paper spray quantification using stable isotope internal standards

Method for MS/MS for monitoring 33 selected metabolites in 0.47 min of analysis

CompoundLOD

(ng/mL)*

CSF (ng/mL) **

CompoundLOD

(ng/mL)

CSF (ng/mL)

Alanine 2 3,300 Lysine 4 28,000Arginine 2 20,200 Methionine 0.3 6,000

Asparagine 13 5,000 Phenylalanin

e0.2 3,000

Aspartic Acid

4 3,000 Proline 4 4,000

Glutamic Acid

0.2 33,000 Serine 4 42,000

Glutamine 6 444,000 Threonine 9 28,000Glycine 5 600 Tryptophan 4 10,000Histidine 4 12,000 Tyrosine 1 10,000

Iso/Leucine 0.48,000/19,00

0 Valine 0.3 24,000

Reactive paper spray for more sensitivity

*in ng/mL or ppb; detection in water solutions (no salts present); ** CSF concentration in (in ppb) based on

literature reports.

Derivatization of the primary amine group on amino acids by acetone forming a Schiff base

Ryan Espy

PAPER SPRAY


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Reactive extraction spray. 18 amino acids can be monitored by MS/MS at CSF concentrations

120 140 160 180 200 220 240m/z

0

20

40

60

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100

0

20

40

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Re

lativ

e A

bu

nd

an

ce

129.15

115.01

229.21157.37143.16

178.87 221.02193.08117.25 236.14 242.86201.06 213.04153.17 183.07245.87

172.21

206.03

222.84158.21 184.05129.22

196.09116.13

NL: 8.81E4blank_acetone_extraction

AV: 15 T: + p ESI Full cnl 46.000 [80.070-300.000]

NL: 5.43E5ACSF 1X_acetone_extraction

AV: 25 T: + p ESI Full cnl 46.000 [80.070-300.000]

glycinealanine Asparagine

histidine

iso/leucine phenyalanine

tyrosinevaline

Christina Ferreira and Yue Ren

Ren et al., 2013. Analytical Methods, 5, 6686-6692.

Some amino acids present neutral loss of 46 after acetone reaction and can be detected by NL scanning

Parallels between embryo and cancer cell metabolism

Energy metabolism: increased glycolysis. Amino acid metabolism: increased glutaminolysis.

Increased fatty acid metabolism: beta-oxidation and de novo synthesis of fatty acids. Parallels in regulation pathways: pyruvate kinase isoform M2 (PKM2) active in cancer cells

and in embryos.Smith and Sturmey, Biochem. Soc. Trans., 41:664-9 (2013).

C.R. Ferreira et al. J Mass Spectrom (2012)C.R. Ferreira et al. Anal. Bioanal. Chem. (2012)Gonzalez-Serrano et al. PLoS One. 2013 Sep 20;8(9):e74981

Mouse or bovine embryos are

washed to remove culture media

Embryos are individually placed onto glass slides

Matured_1to10_Niemann_PL_070312 #103-138RT:3.14-4.20AV:36 SB:140.07-0.46NL:3.53E2T:FTMS - p ESI Full ms [150.00-1000.00]

200 300 400 500 600 700 800 900 1000m/z

0

10

20

30

40

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70

80

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100

Rel

ativ

e A

bund

ance

821.5663

885.5501

181.9372

804.5761

786.5291

742.5393 861.5498

700.5286

288.8971

266.9151906.5437394.0259

920.7562373.8744 479.0038

309.0485

669.1102584.1326506.2885 991.5199

Immature_1to10_Niemann_TAG_070312 #61-73RT:1.28-1.49AV:13 SB:7 0.03-0.14NL:9.57E3T:FTMS + p ESI Full ms [600.00-1200.00]

600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

963.6562

688.1884937.6406

989.6719625.3921

738.5221669.4184

713.4444

1015.6879645.4909 757.4709801.4969 866.4817 1041.7269909.6478 1085.75341134.54881173.8081

DESI-MS analysis

Positive and negative ion mode

free fatty acids, cholesterol esters,

phospholipids, tryacylglycerols and

ubiquinone

High resolution DESI-MS mass spectra of a bovine oocyte (egg) and a blastocyst in the positive ion mode. Abbreviations: TAG – triacylglycerols, Chol ester – cholesterol ester.

ubiquinone ion (of m/z 1140.4, Ag2NO3 adduct) is more abundant in

bovine blastocysts compared to immature oocytes.

Principal component analysis of DESI-MS lipid profiles from control mammary epithelial cells (green circles) and cells genetically modified to express CSCs properties.

ubiquinone adducts appear as major contributors for the differentiation of mouse cells genetically modified to become cancer stem cells (CSCs).

Valentina Pirro. Collaboration with Alice Chang (Purdue University)

DESI-MSUbiquinone as a hallmark in embryo development and cancer stem cells

Bovine immature oocyte

blastocyst produced in vivo

R. Graham Cooks, Cedric D’hue, Allison Dill, Demian Ifa,

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