1

Nuclear Magnetic Resonance (NMR) Spectroscopy

G. A. Nagana GowdaNorthwest Metabolomics Research CenterUniversity of Washington, Seattle, WA

MetabolitesCPMGLactate

AlanineAcetateAcetone

Glutamate

GlutamineThreonine

and -Glucose

Creatinine

CholineValine

EthanolLeucine

Lactate

Tyrosine

Histidine

Formate Tyrosine

Histidine

Protein + Metabolites

1.01.52.02.53.03.54.0 ppm

6.57.07.58.08.59.09.5 ppm

NOESYPR

Blood Based Metabolomics1D NMR

2

• Far fewer number of metabolites ~ 30 or less

• Suppression or attenuation of metabolite peaks due to protein binding

Limitations of Intact Blood serum/plasma Analysis

MetabolitesCPMGLactate

AlanineAcetateAcetone

Glutamate

GlutamineThreonine

and -Glucose

Creatinine

CholineValine

EthanolLeucine

Lactate

Tyrosine

Histidine

Formate Tyrosine

Histidine

Human serum/plasma NMRAlternative Blood Analysis Methods

Ultrafiltration

Precipitation

3

7.07.27.47.67.88.08.28.4 ppm

CPMG

Ultrafiltration

Precipitation

Formate

3-Methylhistidine

1-Methylhistidine

1-Methylhistidine

Histidine

Tyrosine

TyrosineHistidine

Tryptophan

Benzoate

Uridine

Phenylalanine

Tryptophan

(a)

(b)

(c)

Expanding the Limits of Human Blood Metabolite Quantitation Using NMRComparison of Blood Metabolite Analysis Methods

Anal Chem. 2014 Jun 3;86(11):5433-40

Metabolites Extraction using protein precipitation:

Add methanol 2:1

Precipitate protein

Centrifuge, remove protein precipitateDry the supernatant and reconstitute in water

Filtration using molecular weight cut-off filters (3kDa)

Extracted samples can be used for both MS and NMR analyses

Suppression: Physically removing large molecules

Anal Chem. 2015 Jan 6;87(1):706-15

4

• Nearly 70 metabolites identified and quantitated in human blood plasma

Identification and Quantitation of Blood Metabolites

Anal Chem. 2015 Jan 6;87(1):706-15

Whole Blood Metabolomics using NMR

Anal Chem. 2017 Apr 18;89(8):4620-4627

NAD+

NADP+

NADPH

NADH

ATP

AMP

ADP

NAD+

NA

DP

+

NAD+

NAD+

NAD+

NAD+

NADH

NADPH

ATP/ADP/AMP

AMP

ATP ADP

NA

DH

NA

DP

H

NA

DH

/NA

DP

H

NA

DP

+

NA

DP

+

NA

DP

+

NA

DP

+

• Nearly 80 metabolites identified and quantitated in whole human blood

5

02468

10121416182022 2:1 MeOH:Serum

3:1 MeOH:Serum4:1 MeOH:Serum

0102030405060708090

100

0

50

100

150

200

250

300

350

400

450

0102030405060708090

100

02468

10121416182022

2:1 ACN:Serum3:1 ACN:Serum4:1 ACN:Serum

0

50

100

150

200

250

300

350

400

450

Anal Chem. 2015 Jan 6;87(1):706-15

Con

cent

ratio

n (u

M)

Blood metabolites extraction : Choice of solvent

Acetonitrile not good for

protein precipitation

7.27.47.67.88.08.28.48.68.89.09.2 ppm

NAD+

NA

DP

+ NAD+

NAD+

NAD+

NAD+

NADH

NADPH

ATP/ADP/AMP

AMP

ATP

ADP

NA

DH

NA

DP

H

NA

DH

/NA

DP

H

NA

DP

+

NA

DP

+

NA

DP

+

NA

DP

+

8.458.508.558.60 ppm

NAD+

NADP+

NADPH

NADHATP

AMPADP

9 8 7 6 5 4 3 2 1 ppm

Anal Chem. 2016 May 3;88(9):4817-24.

Coenzymes and Antioxidants Analysis in Tissue in one step 

Many coenzymes are unstableChallenging for MS

6

8.458.508.558.60 ppm

AMP

ATP

NADPHNADH

NAD+

NADP+

Simultaneous Analysis of Coenzymes

ng/

mg

tiss

ue

Mice hearts

0

100

200

300

400

500

n=6

8.458.508.558.60 ppm

ATP

AMP

ADP

ATP

NAD+

NADH NADPH NADP+

(a)

(b)

(c)

(d)

Sensitivity to Harvesting/Extraction

Mouse Heart

MeOH-Water

MeOH-CHCl3, in vivo

MeOH-CHCl3, perfusi

MeOH-CHCl3

NADH NADPH

NAD+

NADP+AMP

7

Urine NMR: Sample Preparation

Urine - Intact samples

No preparation required except adding buffer to stabilize pH

About 0.1% NaN3 added to prevent bacterial growth

NATURE PROTOCOLS, 2(11), 2692-2703 (2007)

September 2013 | Volume 8 | Issue 9 | e73076

More number of metabolites detected by NMR than MS

8

Magic angle spinning for intact cells and tissue samples

54.7o Magic Angle

Preparation of Cells and Tissue for NMR

Nuclei interact with other nuclei within the molecule and between molecules

Formate

Acetate

Fumarate

Succinate

Butyrate

Propionate

Short Chain Fatty Acids Analysis using 1H NMR

Pentatonicacid

Hexanoicacid

9

10 9 8 7 6 5 4 3 2 1 0 ppm

Metabolite identification in salty samples (3% salt)

Formate

GlycerolAcetate

1H NMR at 800 MHz

Beta-hydroxybutyrate

lactate

Distinguishing Isomeric Compounds : 13C NMR

Conjugated Linoleic acid (CLA)

10

31P NMR

NMR of isolated organs

J Vis Exp. 2010; (42): 2069.

Mouse heart

NMR of rodents

11

NMR of Animals

NMR of Humans

1H NMR

31P NMR

Functional Imaging

Angiography

Imaging

12

Sample Preparation

Thaw frozen sample

(1) Intact sample- Use directly for NMR

(2) Processed sample- ultrafiltration, protein precipitation (drying)

(3) Mix/dissolve with D2O solvent/buffer(TSP/DSS)

(4) Run NMR

NMR Data Pre‐Processing

Raw data: FID (free induction decay)

1H NMR13C NMR 31P NMR

etc.

13

Apply window functionSmoothening or resolution enhancement

• exponential• gausssian• sine• squared sine• etc

FT (Fourier transformation)

14

Phase correction: After

Baseline correction: Before

15

Baseline correction: After

calibration (peak alignment): Before

16

calibration (peak alignment): After

Binning

17

Peak integration – relative quantitation

Absolute quantitation

18

Angew. Chem. Int. Ed. 10.1002/anie.201804736