1

Using Mixed Isotope Labeling for Systems Biology

Shane LamosVermont Genetics Network Annual

RetreatAugust 7, 2013

2

Systems Biology -Seeks to discover the link between molecules

and physiology

Top Down – “omics”, phenomenon based

Bottom Up – mechanism based e.g. enzyme kinetics

3

Systems Biology – Top Down

4

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

5

H

H

Isotope Dilution vs Isotope Labeling

Experimental 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

OH3N 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

LHN

OH

O

HN

OH

O

NH

LHN

OH

O

NH

H

6

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

nsit

y

X Y

Y

X

yx

yx yx

yx

yx

yx

7

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

8

Isobaric Cholamines

H2NN

CH3

CH3

CH3

H2NN

CD3

CD3

CD3

NH2

NH2

M + 0 M + 9

NH2

NH21. (Boc)2O

2. CX3I, KHCO3, MeOH NHN

O

O X3C CX3

CX3H2N

N

X3C CX3

CX3

Acetyl Chloride

Methanol

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

9

Enhanced Detection of Labeled Metabolites

Valine, Asparagine, Glutamic Acid, Tryptophan

• Ionizability of Analyte is Enhanced

10

365.

3929

366.

3965

374.

4504

375.

4540

+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)

11

Model Egg Labeling with Cholamine

Sample A Sample B

Limits of Detection (LOD) = 10 – 30 fmol

12

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

nsit

y

X Y

Y

X

yx

yx

yx

yx

yx yx

13

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 (O

live

Oil

and

CLA

/ S

aflo

wer

)

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

SCD-1

CLA (18:2)

14

Quantification of Fatty Acids From Other Chicken Egg Diets

15

Multiplexing

Sample A

Sample C

Light Isotope Label

Heavy Isotope Label

yx

yx yx

yx

Sample B

yx

yx

MediumIsotope Label

y

xy

yxx

m/zInte

nsit

y X

X

X

16

Multiplexing Isobaric Cholamines

NH2

N

CH3

CH3

CH3

NH2

N

CH3

CH3

CD3

NH2

N

CD3

CD3

CD3

H2NNH2

M + 0 M + 9

M + 3

NH2

N

1. (Boc)2O

2. CD3I, KHCO3, MeOHNH

NO

O D3C

H2NN

D3C

Acetyl Chloride

Methanol

Cholamine M + 3

17

Simulated Multiplexed Labeling

Sample A Sample B Sample C

18

Multiplex LabelingCaged vs Cage Free

19

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

H2NNH2

M + 0 M + 9

M + 3 M + 6 M + 12

20

Five-Plex Isobaric CholaminesNH2

NH21. (Boc)2O

2. CD2HI, KHCO3, MeOH NHN

O

OHD2C CD2H

CD2HH2N

N

HD2C CD2H

CD2H

Acetyl Chloride

Methanol

Cholamine M + 6

NH2

NH21. (Boc)2O

2. 13CD3I, KHCO3, MeOH NHN

O

OD313C 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

21

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

NH2N

CX3CX3

CX3

22

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

H2NN

H3CCH3

CH3

Cholamine N,N-dimethylaminoputrescine (DMP)

H2NN

CH3

CH3

23

Synthesis of DMP and DMP + 6Non-Isotopic and Isotopic Putrescine (13C0 and 13C4)

Non-Isotopic and Isotopic Formaldehyde (H212CO and

H213CO)

DMP {M + 0, and M + 6}

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

Investigating coupling conditions for precision and accuracyAlso using DMP for enhanced ETD Top-down Proteomics

H2NNH2 1. AcCl (1 eq.), MeOH

2. (Boc)2O

**

* * NH

NH2**

* *

Boc H2*CO (2 eq.), NaCNBH3

NH

N**

* *

Boc

*

*

1 M HCl

H2NN**

* *

*

*

24

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 ‘15Support:Vermont Genetics NetworkSaint Michaels College Gianni Fund and VPAA Fund

25

Saint Michael’s College