Isotope application in Cell

& Genetics
Peni KS Mutalib
Medical Physics Department

Content

Definition/ Abbreviation
Sample/ ReagentCell culture
Basic Principle of Instrument
Methods
FluophoreSemiconductorStable
isotope
Software: ICPL Quant, Cytocluse,
CytoQuick, MultiSET, CellQuest, etc.

Same Z , different A: Isotopic

Same A, different Z: Isobaric
Isotope is a same chemical element
with:
Same number of protons but different
number of netrons, or
Same atom number (Z) but different
mass number (A):
Same chemicals characteristic, but
some differences in physics features
In nature, elements are a mixture of
isotopes (except Be, F, and Na)
Elements with an Z greater than that of
lead (82) is a radioisotopes

A
Z

12
6

13
6

235
92

Abbreviation~Stable
Isotope
SILAC, ICAT, iTRAQ in mammalian & yeast cells
Stable Isotope Labelling Amino acids in Cell Culture
Isotope Coded Affinity Tags
Isobaric Tag for Relative and Absolute Quantitation
hESC: human Embryonic Stem Cell
LC/MS -- GC/MS --MS/MSFlow cytometer/MS
MALDI-TOF: Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight
Mass Spectrometer; Source Nitrogen laser: 337 nm for
metabolomics, peptides & protein, synthetic polymere
SIRMS: Stable Isotope Ratio Monitoring (irm-GC/MS)
FIRMS: Forensic Isotope Ratio Monitoring
LOPIT Localization of Organelle Protein by Isotope Tagging
Transfection Reagents
X-SCID: X-linked Severe Combined Immune Deficiencies

Abbr

ITU band

Frequency

Wavelength

< 3 Hz

> 100,000 km

ELF

330 Hz

100,000 km 10,000 km

SLF

30300 Hz

10,000 km 1000 km

ULF

3003000 Hz

1000 km 100 km

VLF

330 kHz

100 km 10 km

LF

30300 kHz

10 km 1 km

MF

3003000 kHz

1 km 100 m

HF

330 MHz

100 m 10 m

VHF

30300 MHz

10 m 1 m

UHF

3003000 MHz

1 m 100 mm

SHF

10

330 GHz

100 mm 10 mm

EHF

11

30300 GHz

10 mm 1 mm

Above 300 GHz

< 1 mm

HP-MW-FIR-IR-NIR-VIS-UV-X-Ray-Electron-Positron-Gamma Ray Raman

Micromol-nanomol-picomol-femtomolatto-zepto-yocto
NMR-MRIMSFlow cytometry

CD45: Leucocyte
CD3: T Lymphocyte
CD4: Th
CD8: Tk

Fluorophores: PEFITC--PerCP-Cy5.5APC: Phyco Erythrin- Fluorescein

Isothiocyanate-Allophycocyanine
Q-dot semiconductors

Semiconductor
nanotechnology
Nanocrystals are nanometer-scale
(roughly protein-sized) atom
clusters, containing from a few
hundred to a few thousand
atoms of a semiconductor
material (cadmium mixed with
selenium or tellurium), which has
been coated with an additional
semiconductor shell (zinc sulfide)
to improve the optical properties
of the material. These particles
fluoresce in a completely
different way than do traditional
fluorophores, without the
involvement of ->* electronic
transitions.

488 nm 516-655 nm

Nanocrystal for cytometry

Qdot bioconjugate is a generic term to
describe Qdot nanocrystals coupled to
proteins, oligonucleotides, small molecules,
etc., which are used to direct binding of the
quantum dots to targets of interest.
Examples of Qdot bioconjugates include
streptavidin, protein A, and biotin families
of conjugates. Qdot bioconjugates are
often used as simple replacements for
analogous conventional dye conjugates
when their unique performance
characteristics are required to achieve
optimal results.

Semiconductor and
Semiconductor

Color light

Wave
length

Gallium aluminium arsenide

infrared

880 nm

Gallium aluminium arsenide with smaller red

diameter

645 nm

Alluminium indium gallium phosphate

yellow

595 nm

Gallium phosphate

Green

565 nm

Gallium nitride

Blue

430 nm

Immunofluorescence
Multicolor immunofluorescence
imaging with Qdot secondary
antibody conjugates.
Laminin in a mouse kidney section
was labeled with an anti-laminin
primary antibody and visualized
using green-fluorescent Qdot 565
IgG. PECAM (platelet/endothelial
cell adhesion molecule; CD31) was
labeled with an antiPECAM-1
primary antibody and visualized
using red-fluorescent Qdot 655
IgG. Nuclei were stained with bluefluorescent Hoechst 33342.

Key differences between current cell analysis technologies and those using
stalbe isotopes:
DVS Key Technology/Product Differentiators
Current Technology
Flow cytometry is:
well established
mature
uses fluorophores/quantum dots as detectable
tags
Limitations:
can only identify a small number (<10) of
biomarkers simultaneously
variable signal background, and signal overlap and
distortion, have huge impact on accuracy
relative quantitation

DVS Technology
Elemental flow cytometry is :
a novel bio-analytical tool
uses inorganic elements or stable isotopes as
detection tags
Advantages :
many (up to 100) biomarkers in a single cell
simultaneously
high resolution, with little or no signal distortion,
results in high precision and throughput
absolute quantitation

KG1a
THP-1
BCLQ

20-plex cell surface marker

3 leukemic cell lines

FAB M0
FAB M5
Patient M5a

147
145

Nd

Nd
CD14

142

Yb

174

Sm

HLA-DR

156

CD20

CD4

Gd
Er

166

CD15

Eu

153

CD123

CD64

152

Nd CD117

146

CD3

La CD7

139

10

10

10

10

Er CD56

170

CD19 171Yb

CD45

Pr CD33

CD36

141

HoCD38

CD34

165

CD44

Eu

151

CD13

Nd

144

CD49d
176

Yb

169

Sm

Tb

159

Dy

164

Tm
characteristic of
undifferentiated
cells

Isotope in DNA research

The Meselson
- Stahl Experiment
1958

E. coli bacteria were cultured for several generations in heavy nitrogen (15N normal nitrogen is 14N)
The bacteria incorporated 15N into their nucleotides and thus, their DNA
Meselson and Stahl then transferred the bacteria to a medium containing 14N,
Thus, any DNA that the bacteria synthesized would be lighter than the
"old" DNA made with the 15N medium
The DNA was extracted from the cells and centrifuged in a cesium chloride
density gradient for 20 hours at 40,000rpm.
The DNA migrated to a point that was equivalent to their density.
Results from the parental generation contained only a single high
density band - all DNA molecules contained the "heavy" nitrogen.
DNA taken from the two generations after the switch contained an
intermediate-density band - DNA contained a "heavy" DNA strand from
the parent and a complementary "light" DNA strand.
Density results from generation 3, displayed two bands. They included
an intermediate density band, composed of one parental "heavy" strand
and a new light band, composed of only DNA strands with "light"
nitrogen.
Mixing the first and third generations showed all three types of DNA
molecules - heavy, light and intermediate.

N Labeling of DNA:
Semiconservative

15

CsCl gradient
Ultracentrifuge:
Density different

N Labeling of Proteins
Overexpressed in E coli strain
KRX
15

The KRX strain is suitable for applications

that require efficient 15N incorporation into
target proteins.
These cells can be used for target cloning,
protein expression, screening and protein
folding/aggregation assessment by 15N
HSQC or protein identification/quantitation
by mass spectrometry
Single Step (KRX) Competent Cells eliminate
the need to transfer the expression plasmid
into another expression/labeling strain

Protocol for 15N Labeling of Protein

Using the Single Step (KRX)
Competent Cells
Day I
1. Inoculate a single colony from freshly streaked plate into 5
ml of starter culture
2. Grow at 37oC for ~6-8 h
3. Idem no.1 into 250ml of overnight culture
4. Idem no.2 (< 18 h)
Day 2
5. Harvest by centrifugation at 3.000 rpm for 10 m at 4 oC
6. Resuspend cell pellet in 250 ml (vol equal to overnight culture)
of induction media: supplemented with IX metal mix, with
1g/L of NH4Cl or 15NH4Cl (Cambridge Isotopes)
7. Adapt the cells at 37oC for 30 minutes to 1 h
8. Induce with 0.2% (w/v) rhamnose and 0.4 mM cAMP overnight
at 25oC

Day 3
9. Collect cells by centrifugation at 5.000 rpm for
20 min at 4oC
10. Resuspend cell pellet in 25 ml of Cell Lysis
Reagent with 25ul of RNase-Free DNase and
protease inhibitor cocktail in 50 ml
11. Incubate cell resuspension at room
temperature for 20 min
12. Collect the supernatant by centrifugation at
7.500 rpm for 20 min at 4oC
13. Load with Protein Purification Resin, wash of
binding/wash buffer
14. Elute with 10 ml of elution buffer

N Incorporation Efficiency

15

15
Unlabeled
N (Da) %Labelin
(Da)
g
Monster Green 27.346
27.655 94.8
GFP
CRBPII 1
17.012
17.231 97.8
CRBPII 2
17.017
17.224 >99
The molecular weights of purified proteins were
determined by MALDI TOF mass spectrometry. CRBPII
proteins were prepared and evaluated from two separate
cell cultures. Theoretical size of unlabeled Monster
Green GFP is 27.349 Da and of CRBPII is 16.958 Da;
molecular weight of 15N fully substituted Monster Green
GFP is 27.675 Da and CRBPII is 17.153 Da as

Fig. Overlay of MALDITOF mass spectrometry traces of the

unlabeled and 15N-labeled protein. The protein
concentration was ~10 mg/ml; samples were anlyzed by
HT Laboratories Inc.

N-labeled CRBPII expression. Panel A. Proteins from uninduced

cells, induced cell lysate supernatant and eluted from HisLink Resin
were analyzed by SDS-PAGE on 4-20% Tris Glycine gels followed by
Coomassie blue staining. Panel B. 1H-15NHSQC spectrum of ~1 mM
15N CRBPII in 10 mM phosphate buffer with 130 mM NaCl. Data was
recorded at the National Magnetic Resonance Facility on a 750mHz
spectrometer equipped with a cryogenic 1H, 15N, 13C triple-resonance
probe, data points for protein and nitrogen.
15

Nitrogen Laser: 337 nm

MALDI-TOF MS: Matrix-assisted laser
desorption/ionization-Time-of-flight Mass
Spectrometer or other MS could use Nitrogen
laser (337 nm) for seperated/Quantitation
- Metabolomics
- Organelle/Cells
- Peptides
- Protein
- RNA
- DNA

H,

15

N,

13

Radioaktive
nuclide

Energi (MeV) Half-life

(waktu paruh)

H
3
H (beta-)

7.289
14.950

12.33 y

N
13
N (beta+)

0.102
5.346

10 menit

11

C (beta+)
14
C (beta-)

10.650
3.020

20 menit
5730 y

gamma

100 KeV

Penetrasi bbrp
cm

15

Other Protein research

[Determination by 15N tracer kinetics of the half-life of total ...

Based on 15N-tracer techniques important data of the intermediary
protein metabolism can be assessed by compartment analysis. We
calculated the half-life of ...
www.ncbi.nlm.nih.gov/pubmed/4010680 - kaca sing meh padha
Measurement of half-life of human plasma fibrinogen
of glycine into protein in a given time is. obtained from the 15N
enrichment. of glycine incorpo-. rated into protein. We determined.
the half-life ...
ajpendo.physiology.org/cgi/reprint/234/5/E504.pdf - kaca sing meh
padha
Triamcinolone acetonide regulates glucocorticoid-receptor levels ...
... of receptor using media containing 2H, 13C, and 15N-labeled
amino acids. ... Triamcinolone acetonide reduced the half- life in
proportion to the extent of ... 10 nM [3H]triamcinolone acetonide
shortened receptor half-life almost ...
www.jbc.org/cgi/content/abstract/260/1/418 - kaca sing meh padha

Determination by 15N tracer kinetics

of the half-life of total body protein in
premature and mature infants]

Wutzke KD, Heine W, Plath C, Krienke L.

Based on 15N-tracer techniques important data of the
intermediary protein metabolism can be assessed by compartment
analysis. We calculated the half-life of whole body proteins in five
preterm and five full term infants in addition to commonly used
parameters of the protein metabolism e.g. protein synthesis rate,
protein breakdown rate, N-turnover rate, size of metabolic pool,
half-life and reutilization of aminoacid-N and the rate of
endogenous urinary-N. The infants were aged 27 +/- 4 and 31 +/13 days resp. The half-life of whole body proteins were found to be
7.5 +/- 1.8 days in the premature infants and thus significantly
shorter than the 16.0 +/- 3.8 days for the full term infants. The
differences in the half-life of protein as well as protein synthesis
rate and protein breakdown rate reflect the rapid proteinturnover
in premature infants in comparison to full term infants.
PMID: 4010680 [PubMed - indexed for MEDLINE]

Fluorophores/Q-dot/Stable isotopes
w Flowcytometer/ BioNMR/ Mass
Spectrometer
Instead of using optical probes (fluorophores
or quantum dots) to identify biomarkers, DVS
uses elements or stable isotopes. There are
more than 100 available elements and stable
isotopes that are suitable for the application.
The CyTOF uses an application-specific
adaptation of an Inductively Coupled Plasma
Mass Spectrometer (ICP-MS) to detect and
quantify the elemental probes.

Stable isotope: Why Its

Needed
The technology addresses an urgent need for
improvement of detection and
characterization of cancer cells. This provides
improved accuracy and sensitivity of
diagnosis of leukemia and other diseases,
which are often misdiagnosed or diagnosed
too late due to the small number of markers
used in detection. A high number of detected
disease markers allows for personalized
diagnosis and treatment.

PRODUCT APPLICATIONS:
CyTOF
Instrumentation that allows
massively multiplex analysis of
individual cells or beads:
clinical : accurate diagnosis of
disease (or health), stem cell
diseases (e.g., leukemia), riskfree alternative to
amniocentesis
research : cell population
characterization, gene
expression, protein interaction,
simultaneous gene/protein
assay
gene analysis : multiplexed
beads allow 1,000's of
measurements per second

MAXPAR
CyTOFTM instruments reads the stable
isotopic tags attached to Antibodies using
the MAXPAR labeling kits
---Conventional Flow cytometer software
for examination: Cytocluse, cytoQuick,
MultiSET, Cell Quest, for Mac (Macintosh)
Massively multi-parametric cluster analysis
algorithms will provide precase diagnostics
for the clinician
=Mass cytometry offer economy,
simplicity, fast read-out

MAXPAR DNA
Intercalating Kits
The series of MAXPAR DNA intercalating kits
is now available. Each kit contains metalcontaining intercalator, buffers, washes and
instructions. The 1X kit is sufficient to label 800
Million cells (e.g., 800 sample assays of 1 Million
cells/sample); the 5X kit is sufficient to label 4
Billion cells (e.g., 4,000 sample assays of 1
Millions cells/sample). The kits are available with
naturally-abundant Iridium, enriched 191Ir or
enriched 193Ir. The enriched intercalators are
ideal for discrimination of live/dead cells; the
naturally-abundant intercalator is useful for
quantifying DNA of permeabilized or lysed cells.

CDs and intracellular

protein

Lyman, Balmer, Paschen,

Brackett, Pfund Series

ICAT: Isotope Coded Affinity Tags

iTRAQ: isobaric Tag Relative

Absolute Quantitation
The iTRAQ Reagents are the first set of
multiplexed, amine-specific, stable isotope
reagents that can label all peptides in up to
eight different biological samples enabling
simultaneous identification and quantitation,
both relative and absolute, while retaining
important PTM information. There are two
types of iTRAQ Reagents, four plex and
8plex. The new iTRAQ Reagents 8plex are
also available in more economical bulk packs.

Isotope-coded affinity tag

(ICAT)
Linker: Heavy version
will have 13C at *
Light version will have
12
C at *

Isotope-coded affinity tag

(ICAT)

Disadvantages of ICAT
No Cys residues
Cys not accessible for ICAT reagent
Chemical modification of proteins
Tag fragmentation
Meaning of relative quantification
information
Relative Abundance

m/z

SILAC
Stable Isotope Labeling by Amino
Acids in Cell Culture
Based on Mass Spectrometry
Simultaneous: ID and quantify
complex protein mixture

The SILAC Strategy

Mammalian cells cannot synthesize
essential amino acids
If labeled analog of AA supplied
instead of the natural abundance AA,
it will be incorporated into each
newly synthesized protein chain

SILAC ++++
Label incorporated into proteins
during synthesis, encoded into the
proteome
No chemical labeling
No affinity purified steps needed
Method compatible with cell culture
conditions (1 and continuous cell
lines)

EXTRA STEPS

In Comparison
When to use ICAT

When to use SILAC

Can quantitate protein

from non-living
sources, but requires
chemical modification
and affinity steps

Protein populations
from live cells can be
mixed directly after
lysis and then protein
purified.
When you dont want to
lose protein in extra
purification steps

In Comparison
ICAT

SILAC

Labels ~20% tryptic

peptides (2% relative
abundance of cysteine)

Achieves some
decrease in peptide
mixture complexity

Labels more than

tryptic peptides (10%
relative abundance of
leucine)
Does not change
peptide abundances
resulting from a digest.

Quantitative mass
spectrometry reveals a role
for the GTPase Rho1p in
actin organization on the
peroxisome membrane
Marelli et al, 2004

Goals of paper
1. Improve mass spectrometry to
assist in subcellular localization
Discriminate proteins that are components
of the organelle from containments

2. Apply this approach to the yeast

peroxisome

Peroxisome
Ubiquitous organelle
Rids the cell of toxins
Metabolic roles
-oxidation (yeast)

Usually self-replicate
De novo generation?

http://en.wikipedia.org/wiki/Image:Peroxisome.jpg

Samples for ICAT

ICAT I
Enriched
Ti8PM

ICAT II
Enriched

Ti8PP

20KgP:
20,000 g
pellet

11p
Pex

Enriched
for peroxisomes
and mitochondria

pA

Density gradient centrifugation

Samples for ICAT

ICAT I
Enriched
Ti8PM

Yeast strain:
BY4743

ICAT II
Enriched

Ti8PP

Yeast strain
heterozygo
diploid ??

Different yeast strains were used is this a proble

ICAT Results
ICAT I

ICAT II
What are
these ICAT
ratios
dependent
on?

p(E)

the probability of being enriched

ICAT Results
ICAT I

ICAT II
What are these
ICAT ratios
dependent on?
Limitations of:
Mass spec.
Subcellular &
Biochemical
fractionation

p(E)

the probability of being enriched

Candidate
Proteins
PE = peroxisome enrichment
score
Probability of being enriched in the
enriched peroxisomal membrane
fraction as a function of its ICAT ratio

Why might some proteins be

enriched in one ICAT but not
the other?

2d 4d 4d 7d

First
assessment
Can cells use fatty
acids as carbon
source?
Peroxisomal oxidation

Delete gene of interest

Grow on oleic acid/lauric
acid (OL)

Is this a good assay to implicate proteins in peroxis

2d 4d 4d 7d

First
assessment
Can cells use fatty
acids as carbon
source?
Peroxisomal oxidation

Delete gene of interest

Grow on oleic acid/lauric
acid (OL)

Is this a good assay to implicate proteins in peroxis

Genetic redundancy, nonperoxisomal proteins or other subtle

Subcellular distribution of
candidates -- Rho1p
Double labeling
fluorescence confocal
microscopy

Why hasnt anyone seen

Rho1p?

Glucose & Glycero

Plasma
Endomembranes
Oleic acid:
Peroxisomes

Role of Rho1p in peroxisome

function
rho1-2A: temp. sensitive
rho1 deletion mutant
RHO1-2A: rho1-2A with
rho1 reintroduced on a
plasmid

rho1-2A cells have

fewer and
smaller peroxisomes
Why use RHO1-2A instead of
WT strain?

Rho1p interactions
Antibody against TAP-tag
TAP-Pex
GST-Rho1p
GST-Rho1p
TAP-Pex
GST-Rho1p
TAP-Pex

Pex25-Rho1-Actin

WT

rho1

Rho1-Pex25 are
important for
dynamic
assembly/disass
embly of actin
on peroxisomes

Rho1

pex25
Actin

Pex25
Peroxisome

In a nut shell
Without Rho1

With Rh

Pex25

Actin

Rho1

Peroxisome

Pex25

Peroxisome

Actin

Actin binds to peroxisomes

Cannot release

Rho1 plays a role in active

actin organization on
peroxisomes
Needed for peroxisome fission

RNA and RNA Binding

Proteins Participate in Early
Stages of Cell Spreading
through Spreading Initiation
Centers.
Carmen L. de Hoog, Leonard J. Foster, and Matthias Mann
Cell May 28, 2004 117(5):649-62

Focal Adhesions
primary sub-cellular macromolecules
that mediate the regulatory effects of
extracellular matrix (ECM) adhesion
on cell behavior (Chen, 2003).
the mechanical linkages to the ECM,
and as a biochemical signalling hub
to concentrate and direct numerous
signaling proteins at sites of integrin
binding and clustering.

Focal Adhesions
Critical to cellular processes:
Motility
Proliferation
Differentiation
Regulation of Gene Expression
Survival

Sastry and Burridge (2000)

 MRC5 cells are secondary human

lung fibroblasts which undergo
between 60-70 doublings before
senescence.

Goal
To find attachment-specific
interaction partners of specific focal
adhesion components:
Vinculin
Talin
Paxillin
MRC5 lung fibroblast cells

Functional Analysis of the

Attachment Proteome by SILAC
Strategy: highlight attachmentdependent interactions and not just
distinguish specific interactions from
non-specific ones.

Experiment
2 populations of cells grown
in leucine-deficient media

Fig.2

Electron transitions and their resulting wavelengths for Hydrogen.

Lanthanide series

High affinity lanthanide linker

Isotope-based Mass Spectrometry

Direct analysis of dried blood spots utilizi

ng
desorption electrospray
ionization (DESI) mass spectrometry
(Justin M Wiseman, 2010

HPLC-ESI MS/MS: Metabolomics

Cell Biology / Proteomics:

I. I. Stewart, The reproducible acquisition of comparative

liquid chromatography/tandem mass spectrometry data
from complex biological samples. Rapid Communications
in Mass Spectrometry 18 (15):1697-1710, 2004.
D. R. Stover, Differential phosphoprofiles of EGF and
EGFR kinase inhibitor-treated human tumor cells and
mouse xenografts. Clinical Proteomics 1 (1):69-80, 2004.
J. A. Caldwell, Comprehensive comparative proteome
analysis. Proceedings 50th ASMS Conference on Mass
Spectrometry and Allied Topics :57-58, 2002.
B. Larsen, Phosphorylation analysis (phosmap) of
complex protein mixtures. Proceedings 50th ASMS
Conference on Mass Spectrmetry and Allied Topics :165166, 2002.
A. W. Barolet, Administration of exogenous endothelin-1
following vascular balloon injury: Early and late effects
on intimal hyperplasia. Cardiovascular Research 52
(3):468-476, 2001.

RIA:Radio Immuno Assay

Special safety precautions must be observed when
performing RIA methods. Radioactive isotopes are
used by RIA tests to label antigens or antibodies.
Pregnant females should not work in an area where
RIA tests are being performed. Personnel handling
isotope reagents must wear badges which monitor
their exposure to radiation. Special sinks and waste
disposal containers are required for disposal of
radioactive waste. The amount of radioisotope
discarded must be documented for both liquid and
solid waste. Leakage or spills of radioactive reagents
must be measured for radioactivity; the amount of
radiation and containment and disposal processes
must be documented.

STEM CELL

Triple-Encoding SILAC approach

Breast Ca SILAC

Silac drug principle

SILAC sample

Nucleus
fluorescence

Mitochondrion

Possible linkage between Oxidative DNA Damages

and Insulin Resistance (IR) (a No-UCP defence)
Western diet: TNF , iNOS (RNS) apoptosis
IR
Hyperglycaemia
Oxidative
Stress

ATP
ROS
Doxyguanosine

Reduced
Insulin Action
Vicious
Cycle

UCP2 gene
(antiapoptosis)
cells
pancreas off

8oxo,2?
deoxyguanosine
(8-oxodG)

Abnormal gene expression

Somatic Cell Gene Mutation

Conversion of energy
All activities of the body, incl.

Thinking
ATP
Lifting a weight Riding a bicycle
Maintain a constant body temperature
To build a stored energy supply (fat)

Under resting (basal) conditions, bodys energy

is used by:
The
The
The
The

skeletal muscle and the heart (25%)

brain (19%)
kidney (10%)
lever and spleen (27%)

Energy, heat, work

Thermodynamics is defined as the
branch of science that deals with the
relationship between heat and other
forms of energy, such as work.

Power house: Heat and ATP

Harper, 2006

When Beta 3
Adrenoseptor
(Beta3AR) excite, this
send signal to brown
fat which is rich of
thermogenin (Fo
without F1/ATP
synthase) so that no
ATP was formed but
heat. Fo without F1
(thermogenis is also
called uncoupling
protein (UCP). UCP2
was abundant in
omental fat in obese
apple shape person

Metabolic/prediabetic
syndrome

Central obese
Dyslipidemia
Hypertension
Insulin
Resistance
Atherosclerosis
(CHD)

Healthy
Little prone to Diabetes
It is not enough to be
said Diabetes

Beta3AR-UCP2-lemak visceral-Panas

Source of energy (fuel)

Food
Krebs cycle
Anaerobic
CP/ATP
are use in body function
Other external energy sources, such as radiant
solar energy and heat energy from our
surroundings, can help mainain body temperature
but are of no use in body function

Energy is a basic concept of physics, and is of

prime importance in the physics of the body

15N in mitochondria
Real-time study of the urea cycle using 15N n.m.r. in
the isolated ...
The perfused liver was continuously monitored by 15N
n.m.r. spectroscopy at 20.27 MHz for ... mitochondrial
carbamoylphosphate synthetase was rate-limiting. ...
www.pubmedcentral.nih.gov/articlerender.fcgi?
artid=1133080 - kaca sing meh padha
Kasil liyane saka www.pubmedcentral.nih.gov
CiteULike: Mitochondrial Metabolism in Developing
Embryos of ...
10 Nov 2006 ... The resulting flux map shows that
mitochondrial metabolism in these ... [15N]alanine,
(amino)-[15N]glutamine, or (amide)[15N]glutamine, ...
www.citeulike.org/.../9... - 30k Panggonankanggonyimpendatasingwispernahdibuk
ak - kaca sing meh padha

Metabolic Engineering : A window into

cellular metabolism: hepatic ...
Predicted fractional abundance of the
nitrogen mass isotopomers of urea as a
function of the fractional enrichment of 15N
in the mitochondrial ammonia pool ...
linkinghub.elsevier.com/retrieve/pii/S109671
7603000636 - kaca sing meh padha
Kidney International - Abstract of article: The
intensity of ...
Incubation of cultures with [5-15N]
glutamine at pH 7.4 resulted in a ... derived
from enhanced flux through both the
mitochondrial GLDH and PDG pathways. ...
www.nature.com/ki/journal/v45/n4/abs/ki199
4137a.html - kaca sing meh padha

Spectroscope IR
Mol absorpt a specific lambda
(E=hc/lambda) UV move one
electron to an orbital with higher E;
IR make higher vibration amplitudo
atoms which tight each other
(Excited vibration state)
Em:Thermo.
Base line: 100% T

Electromagnetic wave
spectrum

20.000 individual particles/s

Total

Size

Spektroskopi nmr (bagian

hidrokarbon molekul)
Didasarkan pada abs gel radio oleh inti-inti ttt
dlm mol organik, apabila mol ini berada dlm
medan magnet kuat
Inti-inti atom unsur-unsur dibagi: a) mempunyai
spin dan b) tidak mempunyai spin
Inti berspin akan menimbulkan medan magnet
kecil, yg diperikan oleh suatu momen magnetik
nuklir, suatu vektor
1
13
Nuklida ptg yg berspin inti adl
1
H dan
C
6
Sedang 12,6 C dan O tak punya spin
Nuklida berspin menyerap E tak pada radiofrek
yang sama; plg lazim dipelajari dg nmr: proton

Griffin 2004
In the post-genomic era, several profiling tools have been
developed to provide a more comprehensive picture of
tumour development and progression. The global
analysis of metabolites, such as by mass spectrometry
and high-resolution 1H nuclear magnetic resonance
spectroscopy, can be used to define the metabolic
phenotype of cells, tissues or organisms. These
'metabolomic' approaches are providing important
information about tumorigenesis, revealing new
therapeutic targets and will be an important component
of automated diagnosis.

Resonancy
When the frequency of the pulsed radio beam
matches the frequncy of the hydrogen atom, which
induces the protons to flip, this is called the
Resonance Frequency
Resonance in NMR is caused by the absorption of EM radiation in the
radio frequency, by protons in magnetic field (Ho) which then flip.
Flip only happen when the radio wave frequency is suitable. With
strength Ho, the frequency must be more higher (E>)
Flipped proton is also called resonanced proton
When unflipped, there are E that emmited (signal for detector)
computerized curve

Riwayat gelombang radio

diabsorbsi inti atom
1945: Inti atom melalui nuclear spin
mengabsorbsi gelombang radio dari
frekuensi tertentu ketika ditempatkan
dalam sebuah medan magnet kuat
Bbrp th sblmnya: frekuensi rensonansi inti
atom tgtg tak hanya pada 1) kekuatan
medan magnet dan 2) tipe atom, tetapi
juga pada 3) lingkungan kimia atom.
Nuclear spins dari berbagai inti dapat
saling mempengaruhi, memproduksi
struktur-2 halus yaitu sejumlah puncak
dalam spektrum NMR

Bagian utama dari spektrometer NMR

sampel
Generator frekuensi radio
Detektor frekuensi
radio

Puncak ttt utk atom ttt

1966: Sensitivitas yg tadinya kecil dapat
ditingkatkan secara dramatik jika
pengganti berbagai frekuensi pendek
dipakai pulsa intens frek radio untuk
memberi paparan pada sampel
1970: perkembangan cara menentukan 2
inti berdekatan satu dengan yang lain (2
atom berikatan/molekul) sukses pada mol
relatif kecil tapi gagal pada yg lebih besar
karena sulit membedakan resonansi dari
inti atom (spektrum NMR seperti
lapangan.lawn rumput-beribu puncak
shg tak mungkin mengenali puncak mana
utk atom mana terpecahkan oleh Kurt
Wuthrich

Jodoh tiap signal NMR dengan

proton dalam makromolekul
= sequential assignment

1985 Wthrich: Jarak antara sejumlah besar inti hidrogen dan memakai
informasi ini dengan metode matematik bedasarkan geometri jarak untuk
menghitung struktur 3D untuk molekul. Saat ini 15-20% dari ribuan
struktur protein telah ditentukan dg NMR. Yg lain dg kristalografi sinarX, beberapa dg difraksi elektron atau difraksi neutron

Area aplikasi guna NMR

pada makromolekul
Penentuan struktur, NMR dg komplemen kristalografi sinar-X
Keunggulan NMR bahkan untuk bagian molekul trak
terstruktur dan sangat mobil dalam kondisi fisiologis
Struktur protein prion (mad cow: Nobel dlm ilmu kedok 1997
utk Stanley Prusiner)
Struktur dan dinamika makromol DNA & RNA
Dlm industri farmasi: makromol yg jadi target molekul bagi
obat baru. Mol kecil obat baru berikatan dg mol besar mol
besar umumnya berubah. Penting bagi skrining sejumlah
calon obat baru di fase awal perkembangan obat baru.