Metabolomics and Functional

Proteomics

Major Issue with Label-free Quantitative

Metabolomics

Ion suppression

• Signal intensity of an analyte in MS depends on its concentration and

ionization efficiency. Ionization efficiency depends on other components

in the matrix

Solution:• The use of isotope coded standards

– Synthesizing the requisite 13C-coded internal standard can be difficultand expensive.

• Stable isotope coding strategy by in vivo labeling - cells grow inisotopically different media

– Method is not applicable to human and animal studies

• Stable isotope coding strategy by in vitro labeling

Department of Biochemistry

Group Specific Internal Standard

Technology (GSIST)

• GSIST is post-biosynthetic labeling strategy

• GSIST targets specific group in molecules (e.g.primary amines or carboxylic group)

• Components from control samples andexperimental samples are derivatized with twodifferent labeling agents that are chemicallyidentical but isotopically distinct

• After mixing these derivatized components, eachcomponent from control sample serves asinternal standards for determining the relativeconcentration of the chemically identicalcomponent in experimental sample

NH2-AA + NHS-CO-CH3 CH3-CO-NH-AA

NH2-AA + NHS-CO-CD3 CD3-CO-NH-AA

Control Sample

Diseased/Expt. Sample

RPC-ESI-MS

AA from control sample labeled with light form

AA from experimental sample labeled with heavy form

m/z

Department of Biochemistry

Challenge – Lack of Common Functional

Group

Derivatization and LC-MS conditions:

• 300-fold excess of aniline at pH 4.5.• Room temperature for 2 hours• Column: Zorbax C8• Ion pairing reagent concentration: 5 mM TBA• Mobile phase pH 5.0• Elution gradient: 5-70% ACN• ESI – negative mode

Most intermediates contains:

Aldehyde

Carboxyl

Phosphate

Department of Biochemistry

Absolute Quantification - Labeling

Schema

Sample Standards

Combine

Light & Heavy

LC-MS

Light Isotope

Labeling (L)

Heavy Isotope

Labeling (H)

Absolute quantification

based on doublet ratio

Schema 1

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

Inte

nsity, cp

s

1

4

2

3

6

5

7

8

9

11

1210

13

1415

16

17

22

18

1921

20

23

24

27

25

26

28

29

30

32

31 33

D-ribulose-mono-aniline

250 255 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 345 350m/z, amu

0.02000.04000.06000.08000.01.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e4

Inten

sity, cou

nts

304.0592 310.0793

320 325 330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420m/z,amu

0.02000.04000.06000.08000.01.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e43.8e44.0e44.2e44.4e44.6e44.7e4

Inten

sity, cou

nts

379.068 391.148

355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450m/z, amu

0200400600800

100012001400160018002000220024002600280030003200340036003800400042004384

Inten

sity, cou

nts

434.222416.110

a

b c d

b/ D-ribulose 5-phosphate-momo-aniline c/ D-ribose 5-phosphate-bis-paniline d/ citrate tri-aniline

Department of Biochemistry

Epilepsy

• Recurring unprovoked (????) seizures - uncontrolled firing of neurons

• Present in approximately 2% of the population

Causes• Genetic: the direct result of a known or presumed genetic defect(s) in

which seizures are the core symptom of the disorder; e.g., SCN1A gene has been demonstrated to be associated with Epilepsy.

• Structural/metabolic: specific structural or metabolic disorder that has been proven in various studies to be associated with increased risk of epilepsy, The cause can be acquired (e.g., trauma, stroke, infection), genetic (e.g., malformations of cortical development) or both (e.g., West syndrome).

• Unknown cause: This means that the underlying cause is yet unknown. Epilepsy might be presenting because of either some genetic defect or as a result of disorder that is not yet recognized.

Treatment of Epilepsy

• Three main treatments

– Anticonvulsant Drugs

• Topamax, Phenobarbital, benzodiazepines, Dilantin

– Ketogenic Diet

• High fat low sugar diet

– Surgery

• Localized seizure onset zones

Lipid & Energy Metabolism:

Ketogenic “High-Fat “ Diet

TCA

Glycolysis

PPP

AcylCoA

FA

NADPH

ATP

ATP

Lactate

Citrate

Ongoing Studies

Mouse Model of Temporal

Lobe Epilepsy

Kcna1 -/-

Generalized

hippocampal/cortical

seizures

Approx 7 per day

Human Epileptic Brain

Tissue

• Onset

• Irritative

• Silent

Our Targets

• Energy/Lipid Metabolism

• Protein Alternation/Damage

Metabolite Analysis

Lipids

• Global Profiles

• AcylCoA

• Fatty Acid Amides

Amino Acids

• Glu, Asp, Gln, Asn

Energy Metabolism

• ATP, ADP, AMP, cAMP, Adenosine, NADH

• TCA, PPP, Glycolysis, Lactate, Cytosolic Citrate

• NADPH, GSH/GSSG

Amino Acids-aTRAQ

Absciex website

Amino Acids

Summary

• No apparent difference between onset and quiet zones

• Irritative has lower amino acid levels overall

• Signaling molecules remain constant

• Serine: important molecule associated with seizures– 3-Phosphoglycerate dehydrogenase

deficiency is a severe disorder affecting the central nervous system. Patients present with congenital microcephaly, severe psychomotor retardation, and seizures.

TCA

Glycolysis

PPP

AcylCoA

FA

NADPH

ATP

ATP

Lactate

Citrate

Serine biosynthesis

Energy Metabolism

Human Tissue

• Tissue collection prevent ATP and other molecules determination

• Molecules are degraded

Mouse Model

Differential and Functional

Proteomics

Transcription Translation

Protein activity

PTM Protein complex

Misfolding

Proteomics – Experimental Approach

Differential Proteomics

Protein ID FC O/Q p Value Acession Protein

11 1.212187 0.060913 P08559 Pyruvate dehydrogenase E1 component subunit alpha

37 1.290573 0.030006 Q8N945 PRELI domain-containing protein 2

173 1.321941 0.016407 Q6P587 Acylpyruvase FAHD1, mitochondrial

51 1.316746 0.033578 Q96JH7 Deubiquitinating protein VCIP135

52 1.34685 0.011562 A6NHL2 Tubulin alpha chain-like 3

9 2.612303 0.037366 P08729 Keratin, type II cytoskeletal 7

263 1.232938 0.040662 Q96MP5 Zinc finger SWIM domain-containing protein 3

245 0.825042 0.047108 P59768 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

73 0.690256 0.080468 Q6IQ20 N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D

Functional Proteomics -

Protein Carbonylation

From J Proteome Res. 2010 August 6; 9(8): 3766–3780.

Lysine Modification Competition

Sample preparation

Biotinylation of carbonylated proteins

Affinity selection with avidin

nanoRPLC-MS/MS (ion trap)

Peptide/Protein ID

Trypsin digestion

Strategy for identification of

carbonylated proteins

+ O = C - protein

N = CH - protein

Identification of

Oxidized Amino Acids

Peroxiredoxin (TSA) Thioredoxin (TRX)

Protein Based Strategy for Quantification

• Hydrazines react with aldehydes/ketones

– Hydrazine Cy3/Cy5

• Combine 2 samples

– Control and Sample (disease/treatment)

2DIGE for Carbonylated

Proteins

Spot Anova (p) pI MW (kDa) Oxidation Increase Spot Anova (p) pI MW (kDa) Oxidation Increase1 0.011 7.22 25 3.3 40 0.182 8.87 46 1.7

2 0.015 6.15 32 3.1 41 0.125 7.08 60 1.6

3 0.012 6.51 23 3.0 42 0.149 8.28 22 1.6

4 0.025 6.42 16 2.6 43 0.115 7.44 22 1.6

5 0.030 6.43 16 2.0 44 0.134 9.18 22 1.6

6 0.047 6.66 16 1.9 45 0.190 7.07 59 1.6

7 0.023 6.13 22 1.8 46 0.166 6.96 58 1.5

8 0.026 5.97 33 1.7 47 0.197 4.35 14 1.5

9 0.021 7.3 25 1.7 48 0.129 8.08 20 1.5

10 0.032 6.09 25 1.5 49 0.222 8.25 32 1.5

11 0.039 5.78 25 1.5 50 0.446 9.48 54 2.3

12 0.037 5.16 88 1.5 51 0.442 7.1 17 2.2

13 0.026 5.79 56 1.4 52 0.404 5.22 1.9

14 0.088 9.18 34 2.7 53 0.326 6.85 74 1.9

15 0.060 6.4 22 2.0 54 0.314 5.64 83 1.8

16 0.067 6.24 39 1.7 55 0.459 5.64 117 1.8

17 0.076 8.94 42 1.7 56 0.303 4.96 42 1.7

18 0.068 6.86 63 1.7 57 0.315 9.24 26 1.7

19 0.099 5.88 56 1.7 58 0.293 4.83 58 1.7

20 0.083 5.55 40 1.7 59 0.370 6.68 74 1.6

21 0.073 5.73 35 1.6 60 0.301 5.65 144 1.6

22 0.081 6.81 63 1.5 61 0.433 9.33 33 1.6

23 0.071 6.62 23 1.5 62 0.284 7.16 47 1.6

24 0.187 7.1 17 3.0 63 0.268 4.33 15 1.6

25 0.148 4.96 57 2.6 64 0.282 5.63 25 1.6

26 0.223 9.26 26 2.5 65 0.430 5.49 34 1.5

27 0.112 5.95 32 2.1 66 0.273 8.87 42 1.5

28 0.104 9.01 42 2.1 67 0.295 6.19 16 1.5

29 0.201 6.75 74 2.0 68 0.281 9.26 60 1.5

30 0.190 8.95 34 2.0 69 0.591 4.92 49 2.8

31 0.230 5.59 83 1.9 70 0.866 9.48 45 1.9

32 0.197 3.9 69 1.9 71 0.567 4.08 245 1.9

33 0.121 9.29 42 1.8 72 0.549 7.51 99 1.8

34 0.131 6.85 22 1.8 73 0.687 9.46 60 1.8

35 0.252 5.64 182 1.8 74 0.846 9.52 34 1.7

36 0.185 5.85 51 1.8 75 0.918 9.48 42 1.6

37 0.237 6.47 61 1.7 76 0.581 6.39 73 1.6

38 0.203 9.29 46 1.7 77 0.887 4.58 241 1.6

39 0.126 9.02 39 1.7 78 0.544 7.27 121 1.6

Silent Zone Spot with ≥ 1.5 Fold Oxidation than Pool

Legendp-value ≤ 0.05

p-value > 0.05, ≤ 0.1

p-value > 0.1, < 0.25

p-value > 0.25, < 0.5

p-value > 0.5Legend

Affected Proteins

• VDAC1

– Elevated levels in refractory epilepsy

– Adenine transport (ATP from mitochondria)

– Interacts with bcl-2 family proteins

– Mitochondrial permeability-CytoC

– At the plasma membrane

• Increased cellular NADH=closure

• Modulate Aβ induced cell death

• Redox homeostasis

• UCHL1

– Mutations associated with Parkinson’s Disease

– 3.2% prevalance of comorbidity

• Creatine kinase

– Pretreatment with creatine reduces PTZ induced seizures

• HSP70

– Full coverage in MASCOT

• Actin

– Low score, only 2 peptides

GAPDH

GAPDH – Key Energy Metabolism

Enzyme

Fructose-6-phosphate / 0.49 / 2.23 / B D-6-Phospho-glucono-δ-lactone / 0.48 / 4.22 / NS

Fructose 1,6-bisphosphate / 2.88 / 2.04 / B 6-Phospho-gluconate / 0.18 / 11.85 / B

Ribulose-5-phosphate / 0.15 / 7.31 / BGlyceraldehyde-3-phosphate / 2.44 / 4.40 / B

3-Phospho-D-glycerol / 0.55 / 2.23 / B

Ribose-5-phosphate / 1.72 / 12.94 / B Xylulose-5-phosphate / 0.34 /8.37 / B

Sedoheptulose-7-phosphate / NS

Erythrose-4-phosphate / 0.20 / 3.32 / B

Glycolysis Pentose Phosphate Pathway

Dihydroxy-acetone-phosphate /

0.54 / 3.49 / B

ATPADP

NAD NADH

3-Phosphoglycerate / 1.10 / 0.86 / B

2-Phosphoglycerate / NS

Phosphoenolpyruvate / 0.13 / 0.13 / B

Pyruvate / ND

Acetyl CoA / NN

ATP

ADPATP

NADHNAD

TCA Cycle

Isocitrate / 5.46 / 2.07 / B

α-Ketoglutarate / 2.24 / 3.60 / B

Succinyl CoA / NN

Succinate / 11.46 / 2.98 / B

Fumarate / 11.46 / 2.98 / B

Malate / 2.62 / 2.28 /B

Oxaloacetate / 27.10 / 1.96 / B

FAD

NADH

NADH

ATP

ADP

NADPNADPH

NADPNADPH

Glyceraldehyd-3-phosphate / 2.44 / 4.40 / B

Fructose-6-Phosphate / 0.49 / 2.23 / B

Glyceraldehyde-3-phosphate / 2.44 / 4.40 / B

ATPADP

Glucose-6-phosphate / 1.83 / 1.98 / B

Glucose / 75.98 / 5.98 / B

ADP

NADCitrate /

58.27 / 3.72 / B

NAD

AMP / 0.29 / 0.92 / B

ATP / 2.45 / 1.77 / B

ADP / 1.01 / 1.42 / B

Conc. umol/g DW

RSD, % n=5

Quant. meth

GAPDH

Proteomics – Impact on Energy Metabolism

Ex Vivo Indirect Measurement

TCA

Glycolysis

PPP

AcylCoA

FA

NADPH

NADH ATP

NADH

GA

PD

H

Transketolase

G6PDH

Determine NADH and NADPH production:

Brain lysate

Excess NAD and NADP

Excess of individual substrates:

• Glucose 6 phosphate – PPP activity (NADPH)

• Ribulose 5 phosphate – PPP activity of nonoxidative branch (NADH)

• Glyceraldehyde 3 P – Glycolysis and TCA Activities (NADH)

• Pyruvate – TCA Activities (NADH)

Potential Model: Hyper-Sensitive GAPDH ?

TCA

Glycolysis

PPP

AcylCoA

FA

Glucose

Lipids

KD Diet

ATP

NADPH

GA

PD

H

TCA

Glycolysis

PPP

AcylCoA

FA

Glucose

Lipids

KD Diet

ATP

NADPH

GA

PD

H

Pro-convulsant Anti-convulsant

Potential Treatment:

Increase Redox CapacityUse alternative sources for TCA cycle such as TCA intermediates or Acetoacetyl-CoA???

Prelim Data:

High VitC and pyruvate can reduce frequency of seizures

PTZ induced C. elegans Model

siRNA Method• Grow HT115(DE3) with plasmid ON• Plate 500uL on NGM +Carb +IPTG

ON• Place 30 N2 worms at L1/L2 stage

– Currently synchronizing so all start L1

• Grow to adult (approx 60 hours)• Plate worms to test plate

Initial Targets• Lis1 – neuronal development/seizures

(positive control• Fgt1 – glucose transporter• GFP – negative control

Sampling

We dreamed and schemed tobuild tools that will detectmolecules and the biologicalsignatures they bear.

While physicians dream that

these technologies can impact their world.

Preferred Matrix for Diagnostics

and Drug Testing is Plasma not

Blood

Majority of Clinical Tests use Plasma• Clinical range usually ng/mL• Some tests require pg/mL• Latest LC/MS technology only requires a

few microliters of plasma

Plasma Prepared by Centrifugation at Lab Site

Majority of Pharma Tests use Plasma• Dosage range usually ng/mL• FDA still prefers plasma data for IND, NDA• Latest LC/MS technology only requires

one microliter of plasma

For LC/MS a majority of

the plasma sample is

often discarded

Confidential

Spo

t Si

ze25% 50% 70%

Hematocrit

Alternative: Dried Blood

Spot Cards

Introduced 50 years ago

Basically filter paper with printed circles

Each circle requires about 25 µL of blood

Standard punch recovers 3.2 µL of blood

which translates into 1.8 µL of plasma

Performance affected by hematocrit

Hematocrit = volume % of red blood cells, typically

45% but can vary from 20 to 70%

NoviPlex Plasma Separation

Device

Within 3 minutes, a 2.5

uL plasma aliquot is

extracted from whole

blood.

Noviplex Tested with ELISA

Kearney et al. tested 84 patients for thepresence of antibodies to H. pylori in bloodsamples obtained by venipuncture and bythe NoviPlex card. There was a high degreeof correlation of quantitative EIA resultsbetween specimens obtained byvenipuncture as compared to thoseobtained by the NoviPlex card (r=0.98).There was also a high degree of correlationbetween duplicated cards (r=0.996). Thesensitivity and specificity of the NoviPlexdried plasma card were calculated using thevenipuncture specimen as the goldstandard. This revealed a sensitivity of 93 %and a specificity of 100 % in first duplicateand 98% in second duplicate .

Vit. D Derivatization and Selectivity

CH 3

CH3

H

H

HO

HO

CH 3

CH3

H

H

HO

HO

N N O

ON

N+

CH3

CH2

HN N O

ON

N+

CH3

CH2

m/z = 107

m/z = 207

m/z = 149

SecoSETSecosteroid product ions

The derivative portion of the molecules

► contains a quaternary amine

► provides the dominant product ions

► provides the same mass for all vitamin D

analogues

The steroid portion provides

► unique chromatographic retention

► precursor ion masses that differ

Functional group specific derivatization along with enhanced ionization of the

derivative increases selectivity.

Secosterioid Signal Enhancing Tag

Derivatization is

based on a Diels

Alder addition

reaction

Accuracy of Method

NIST-SRM972a L3

LLE100

NIST provided

(measured)

25OHVD2 (ng/ml) 13.650.41 13.35

25OHVD3 (ng/ml) 19.710.64 19.81

Total (ng/ml) 33.361.05 33.16

Accuracy of the LLE kit using the SecoSET derivatization reagent for quantification

was evaluated with a plasma sample (NIST-SRM972a L3) obtained from NIST

(Gaithersburg, MD). Vitamins in plasma sample were derivatized with SecoSET. The

accuracy of the LLE kit was evaluated relative to values provided by NIST and found

to have a % RSD of 0.4% in the total vitamin D concentration between the

measurement and NIST value.

LOD ~10 pg/mL depending on MS sensitivity; LOD of newest triple quads is 1 pg/mL.

Noviplex Samples Stability

Stability of 25OHVD3 in plasma (short-term)

Weeks

0 2 4 6 8 10

% Re

lative

value(

T0=10

0%)

0

20

40

60

80

100

120

140

160

-80

-20

4

RT

37

Stability of 25OHVD3 in plasma (long-term)

Months

0 2 4 6 8 10 12 14

% Rela

tive va

lue (T0

=100%

)

0

20

40

60

80

100

120

140

160-80

-20

4

RT

37

• Deposit blood

onto surface of

the NoviMet

card:

• Test area will

change to red in

30 sec

• Wait 3 min and

peel of the top

layer of the card

• Let collection

disc to air-dry for

15 min

• Transfer

collection disk

into a well of the

96-well plate

(with flat bottom)

• Use plastic tools

to move the disk

and place it onto

the bottom

• Add 10 µL nitric

acid (100%) and

incubate for 2 hrs

at RT

• Add 190 µL water

and incubate for 1

hr at room

temperature

• Add 200 µL

Internal Standard

(100 ppb Ga in

water)

• Inject the samples

directly into ICP-MS

• Collect data for:

Na(23), K(39), Ca(40),

Se(78)

• Other options:

Mg(24), P(31), S(34),

Mn(55), Fe(56/57),

Zn(64), As(75), Cd(111),

Hg(202)

Determination of Na, K, Ca and Se

Levels in Plasma Using NoviMet

Card

Results1

35

–1

50

mM

NormalRange

NoviMetData

ClinicalData

14

3.3

+/-

5.3

mM

14

1 m

M

Na

3.5

–5

.5 m

M

NormalRange

NoviMetData

ClinicalData

5.5

+/-

0.2

mM

5.4

mM

K

2.1

–2

.8 m

M

NormalRange

NoviMetData

ClinicalData

2.5

+/-

0.3

mM

2.4

mM

Ca

0.8

0 –

1.6

0 µ

M

NormalRange

NoviMetData

ClinicalData

0.7

8 +

/-0

.07

µM

N/A

Se

Thank You