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)
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)