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Using Mixed Isotope Labeling for Systems Biology

Shane LamosVermont Genetics Network Annual

RetreatAugust 7, 2013

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Systems Biology -Seeks to discover the link between molecules

and physiology

Top Down – “omics”, phenomenon based

Bottom Up – mechanism based e.g. enzyme kinetics

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Systems Biology – Top Down

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Research Projects Develop new chemical methodologies to

explore biological systems

Mixed Isotope labeling coupled with MS

Proteome• Identification of small molecule protein targets• Enhanced detection of proteins with charge

auxiliaries Metabolome

• Relative quantification of amine metabolites• Relative quantification of fatty acid metabolites• Multiplexed analysis of carboxylic acid

metabolites • Relative quantification of small carboxylic acids

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H

H

Isotope Dilution vs Isotope

LabelingExperimental Metabolites

Internal Standard

Label with Heavy (H) Reagent

Label with Light (L) Reagent

?

H3N OH

O

NH

H3N OH

O H3N OH

O

**

H3N OH

O

H3N OH

O

**

H3N OH

O

NH

Experimental Metabolites

Control MetabolitesH3N OH

O

NH

H3N OH

O

H3N OH

O

NH

H3N OH

O

L

HN

OH

O

L

HN

OH

O

HN

OH

O

NH

L

HN

OH

O

NH

H

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How Does Relative

Quantification of Metabolites

Work?Sample A Sample B

Light Isotope Label

Heavy Isotope Label

Mix Samples

Separate Analytes

Electrospray MS

m/z

Inte

nsi

ty

XY

Y

X

y

x

yx

yx

y

x

y

x

y

x

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Cholamine as an Isotopic Label

for Fatty Acids

• Fixed positive charge enhanced detection in positive ion-mode (MS)

• Isotopically labeled metabolites co-elute

• Light and heavy label signifies carboxylic acid functionality

H2NN

R OH

O

R

O

NH

N

Cholamine

H2NN

H3C

CH3

CH3

H2NN

D3C

CD3

CD3

Light M + 0

Heavy M + 9

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Isobaric Cholamines

H2N

N

CH3

CH3

CH3

H2N

N

CD3

CD3

CD3

NH2

NH2

M + 0 M + 9

NH2

NH21. (Boc)2O

2. CX3I, KHCO3, MeOH NH

N

O

OX3C CX3

CX3H2N

N

X3C CX3

CX3

Acetyl Chloride

Methanol

X = H, Cholamine M + 0X = D, Cholamine M + 9

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Enhanced Detection of Labeled

Metabolites

Valine, Asparagine, Glutamic Acid, Tryptophan

• Ionizability of Analyte is Enhanced

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36

5.3

92

9

36

6.3

96

5

37

4.4

50

4

37

5.4

54

0

+MS, 20.8-21.1min #(2485-2516)

0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens.

364 366 368 370 372 374 376 m/z

Extracted Ion Chromatograms of

Co-Eluting Heavy- and Light-

Labeled Carboxylic Acids

20.5 21.0 21.5 22.0 Time [min]0

1

2

3

4

5

5x10Intens.

EIC 339.356-339.396 +All MS EIC 348.414-348.454 +All MSEIC 363.359-363.399 +All MS EIC 372.416-372.456 +All MSEIC 365.375-365.415 +All MS EIC 374.432-374.472 +All MSEIC 389.377-389.417 +All MS EIC 398.435-398.475 +All MSEIC 413.38-413.42 +All MS EIC 422.438-422.478 +All MSEIC 341.372-341.412 +All MS EIC 350.429-350.469 +All MSEIC 367.39-367.43 +All MS EIC 376.448-376.488 +All MSEIC 391.393-391.433 +All MS EIC 400.451-400.491 +All MSEIC 415.396-415.436 +All MS EIC 424.453-424.493 +All MSEIC 369.407-369.447 +All MS EIC 378.464-378.504 +All MS

16:1

18:2

16:018:1

18:0

20:4

(Fatty Acid 18:2)

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Model Egg Labeling with Cholamine

Sample A Sample B

Limits of Detection (LOD) = 10 – 30 fmol

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Relative Quantification of

Chicken Egg Fatty Acids

Sample A (CLA + Olive Oil)

Sample B (Control)

Light Isotope Label

Heavy Isotope Label

Mix Samples

Separate Analytes

Electrospray MS

m/z

Inte

nsi

ty

XY

Y

X

y

x

yx

y

x

y

x

yx

yx

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Quantification of Fatty Acids

From Chicken Eggs (CLA + OO vs

Control)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

16:1 18:3 18:2 20:4 22:6 16:0 18:1 20:3 22:5 18:0

Fatty Acid

Rat

io (

Oliv

e O

il an

d C

LA /

Saf

low

er)

(Juvenile Egg) 16:0 + 18:0 16:1 + 18:1 (Fertile Egg)

SCD-1

CLA (18:2)

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Quantification of Fatty Acids

From Other Chicken Egg Diets

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Multiplexing

Sample A

Sample C

Light Isotope Label

Heavy Isotope Label

y

x

yx

yx

yx

Sample B

yx

yx

MediumIsotope Label

y

xy

yx

x

m/zInte

nsi

tyX

X

X

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Multiplexing Isobaric Cholamines

NH2

N

CH3

CH3

CH3

NH2

N

CH3

CH3

CD3

NH2

N

CD3

CD3

CD3

H2N

NH2

M + 0 M + 9

M + 3

NH2

N

1. (Boc)2O

2. CD3I, KHCO3, MeOHNH

N

O

OD3C

H2N

N

D3C

Acetyl Chloride

Methanol

Cholamine M + 3

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Simulated Multiplexed Labeling

Sample A Sample B Sample C

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Multiplex LabelingCaged vs Cage Free

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Five-Plex Isobaric Cholamines

NH2

N

CH3

CH3

CH3

NH2

N

CH3

CH3

CD3

NH2

N

CD3

CD3

CD3

NH2

N

CD2HCD2H

CD2H

NH2

N

13CD3

13CD3

13CD3

H2N

NH2

M + 0 M + 9

M + 3 M + 6 M + 12

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Five-Plex Isobaric Cholamines

NH2

NH21. (Boc)2O

2. CD2HI, KHCO3, MeOH NH

N

O

OHD2C CD2H

CD2HH2N

N

HD2C CD2H

CD2H

Acetyl Chloride

Methanol

Cholamine M + 6

NH2

NH21. (Boc)2O

2. 13CD3I, KHCO3, MeOH NH

N

O

OD3

13C 13CD3

13CD3H2N

N

D313C 13CD3

13CD3

Acetyl Chloride

Methanol

Cholamine M + 12

Using a silkworm hyperglycemic model of type II diabetes

Silkworms grown under 5 different diet scenariosMeasuring free fatty acids in hemolymph

simultaneously

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Cholamines as Isotopic Labels

Effective for relative quantification of carboxylic acids

Pre-ionized fatty acids readily quantified in LC-MS

Heavy- and light-labeled analytes co-elute

“Multiplexing’ of carboxylic acid functionality

NH2

N

CX3

CX3

CX3

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Hydrophobic Tag for Small Carboxylic Acids

Many important small carboxylic acid metabolites

Too polar for chromatographic retention labeled with cholamine

Need a more hydrophobic mixed isotope labeling reagent

HO

O

O

OHOH

O

Succinic Acid Acetic Acid

H2N

N

H3C

CH3

CH3

Cholamine N,N-dimethylaminoputrescine (DMP)

H2N

N

CH3

CH3

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Synthesis of DMP and DMP + 6

Non-Isotopic and Isotopic Putrescine (13C0 and 13C4)Non-Isotopic and Isotopic Formaldehyde (H2

12CO and H2

13CO)

DMP {M + 0, and M + 6}

Labeled a series of small carboxylic acids with DMP {0, and 6}

Investigating coupling conditions for precision and accuracy

Also using DMP for enhanced ETD Top-down Proteomics

H2N

NH2 1. AcCl (1 eq.), MeOH

2. (Boc)2O

**

* *NH

NH2**

* *

Boc H2*CO (2 eq.), NaCNBH3

NH

N**

* *

Boc

*

*

1 M HCl

H2N

N**

* *

*

*

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AcknowledgementsCollaborators:

Dr. Ying Wai Lam Director UVM/VGN Proteomics Facility

Dr. Lloyd M. Smith UW-Madison

Group Members:Jon Downey ’09 Andrew Therrien ‘12Zach Eldridge ’10 Christopher Dustin ‘12Katie Summo ’10 Heidi Chapman ‘13Jeff Dukette ’11 Emily Dieter ‘14Derrick Cumberbatch ’11 Wes Cubberley ‘14Gus Torde ’11 *Katie Schutt ‘14

*Sam Drajesk ‘15

Support:Vermont Genetics NetworkSaint Michaels College Gianni Fund and VPAA Fund

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Saint Michael’s College