2.isotope application in cell & genetics10.ppt

7/24/2019 2.Isotope application in Cell & Genetics10.ppt

1/160

Isotope application in Cell

& Genetics

Peni KS Mutalib

Medical Physics Department


2/160

Content

Defnition/ Abbreviation

Sample/ ea!ent"Cell culture

#asic Principle o$ Instrument Methods

%luophoreSemiconductorStable

isotope So$tare' ICP( )uant* Cytocluse*

Cyto)uic+* MultiS,-* Cell)uest* etc.


3/160

Same * di0erent A' IsotopicSame A* di0erent ' Isobaric

Isotope is a same chemical elementith'

Same number o$ protons but di0erentnumber o$ netrons* or

Same atom number 12 but di0erentmass number 1A2'

Same chemicals characteristic* butsome di0erences in physics $eatures

In nature* elements are a mi3ture o$isotopes 1e3cept #e* %* and 4a2

,lements ith an !reater than that o$lead 1562 is a radioisotopes

A

Z

235

92

X

U

C

C

12

6

13

6


4/160

Abbreviation78StableIsotope9

SI(AC* ICA-* i-A) in mammalian & yeast cellsStable Isotope (abellin! Amino acids in Cell Culture

Isotope Coded A:nity -a!s Isobaric -a! $or elative and Absolute )uantitationh,SC' human ,mbryonic Stem Cell

(C/MS ;; GC/MS ;;MS/MS"%lo cytometer/MSMA(DI;-


5/160

Abbr ITUband Frequency Wavelength

B ? = EFF*FFF +m

,(% E ??F = EFF*FFF +m EF*FFF +m

S(% 6 ?F?FF = EF*FFF +m EFFF +mH(% ? ?FF?FFF = EFFF +m EFF +m

(% J ??F += EFF +m EF +m

(% ?F?FF += EF +m E +m

M% L ?FF?FFF += E +m EFF m

% @ ??F M= EFF m EF m

% 5 ?F?FF M= EF m E m

H% ?FF?FFF M= E m EFF mm

S% EF ??F G= EFF mm EF mm

,% EE ?F?FF G= EF mm E mm

Above ?FF G= B E mm

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

Micromol-nanomol-icomol-!emtomol"atto-#eto-yocto

NMR-MRI"MS"Flo$ cytometry
http://www.economicexpert.com/a/International:Telecommunication:Union.htmhttp://www.economicexpert.com/a/Hz.htmhttp://www.economicexpert.com/a/Km.htmhttp://www.economicexpert.com/a/KHz.htmhttp://www.economicexpert.com/a/Character:M.htmhttp://www.economicexpert.com/a/MHz.htmhttp://www.economicexpert.com/a/Mm.htmhttp://www.economicexpert.com/a/GHz.htmhttp://www.economicexpert.com/a/GHz.htmhttp://www.economicexpert.com/a/Mm.htmhttp://www.economicexpert.com/a/MHz.htmhttp://www.economicexpert.com/a/Character:M.htmhttp://www.economicexpert.com/a/KHz.htmhttp://www.economicexpert.com/a/Km.htmhttp://www.economicexpert.com/a/Hz.htmhttp://www.economicexpert.com/a/International:Telecommunication:Union.htm


6/160


7/160

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

Isothiocyanate-Allophycocyanine%-&ot semicon&'ctors

CD45: LeucocyteCD3: T Lyphocyte

CD4: Th

CD!: T"


8/160

Semiconductornanotechnolo!y

4anocrystals are nanometer;scale1rou!hly protein;si=ed2 atomclusters* containin! $rom a $ehundred to a $e thousand

atoms o$ a semiconductormaterial 1cadmium mi3ed ithselenium or tellurium2* hich hasbeen coated ith an additionalsemiconductor shell 1=inc sulfde2to improve the optical propertieso$ the material. -hese particlesNuoresce in a completelydi0erent ay than do traditionalNuorophores* ithout theinvolvement o$ ;O electronictransitions.


9/160


10/160

J55 nm EL;L nm


11/160


12/160


13/160

4anocrystal $or cytometry

)dot bioconQu!ate is a !eneric term todescribe )dot nanocrystals coupled toproteins* oli!onucleotides* small molecules*etc.* hich are used to direct bindin! o$ the

Ruantum dots to tar!ets o$ interest.,3amples o$ )dot bioconQu!ates includestreptavidin* protein A* and biotin $amilieso$ conQu!ates. )dot bioconQu!ates areo$ten used as simple replacements $or

analo!ous conventional dye conQu!ateshen their uniRue per$ormancecharacteristics are reRuired to achieveoptimal results.


14/160


15/160


16/160

Semiconductor and

Semiconductor Color light Wavelength

Gallium aluminium arsenide infrared 880 nm

Gallium aluminium arsenide ith smallerdiameter

red 6!5 nm

"lluminium indium gallium #hos#hate $ello 595 nm

Gallium #hos#hate Green 565 nm

Gallium nitride %lue !30 nm


17/160

ImmunoNuorescence

Multicolor immunofuorescenceimaging with Qdot secondaryantibody conugates!(aminin in a mouse +idney section

as labeled ith an anti;lamininprimary antibody and visuali=edusin! !reen;Nuorescent )dot LI!G. P,CAM 1platelet/endothelialcell adhesion molecule> CD?E2 as

labeled ith an antiP,CAM;Eprimary antibody and visuali=edusin! red;Nuorescent )dot LI!G. 4uclei ere stained ith blue;Nuorescent oechst ???J6.


18/160

(VS )ey *ec+nolo,yPro&'ct (i!!erentiators

#ey $i%%erences &et'een current cell analysis technolo(ies an$ those usin(

stal&e isotopes:

Current Technolo(y

Flo$ cytometry is.

$ell esta/lis+e&

mat're

'ses !l'oro+ores0'ant'm &ots as &etecta/le

ta,s

1imitations.

can only i&enti!y a small n'm/er 23456 o!/iomar7ers sim'ltaneo'sly

8aria/le si,nal /ac7,ro'n&9 an& si,nal o8erla an&

&istortion9 +a8e +',e imact on acc'racy

relati8e 0'antitation

D)* Technolo(y

Elemental !lo$ cytometry is .

a no8el /io-analytical tool

'ses inor,anic elements or sta/le isotoes as

&etection ta,s

:&8anta,es .

many 2' to 4556 /iomar7ers in a sin,le cell

sim'ltaneo'sly+i,+ resol'tion9 $it+ little or no si,nal &istortion9

res'lts in +i,+ recision an& t+ro',+'t

a/sol'te 0'antitation


19/160

+4,*+45$

+4$

+,4/&

+40$

+31La

+,2Er

+4+Pr

+05o

+5+Eu+44

$

+,0/&

+01T

+04Dy

+51T&

+,+/&

+5*

+53Eu

+00Er

+50$

CD+1

CD3

CD04

CD+5

CD2LA-D

CD+4

CD4

CD+3

CD++,

CD,

CD50

CD33

CD3!

CD44CD+3

CD41$

CD34

CD30

CD45

+2 2 +2 + +2 +2 3

)G4a

*HP-4

;C1%

FA6 72

FA6 75

Patient 75a

2-ple8 cell sur%ace ar"er

3 leu"eic cell lines

characteristic o%

un$i%%erentiate$cells


20/160

Isotope in D4A research
http://www.uic.edu/classes/bios/bios100/lecturesf04am/meselson_stahl_experiment.htmhttp://www.uic.edu/classes/bios/bios100/lecturesf04am/meselson_stahl_experiment.htm


21/160

" #tahl $%&eriment

E. colibacteria ere cultured $or several !enerations in heavy nitro!en 1E4 ;normal nitro!en is EJ42 -he bacteria incorporated E4 into their nucleotides and thus* their D4A

Meselson and Stahl then trans$erred the bacteria to a medium containin! EJ4* -hus* any D4A that the bacteria synthesi=ed ould be li!hter than the

ToldT D4A made ith the E4 medium-he D4A as e3tracted $rom the cells and centri$u!ed in a cesium chloride

density !radient $or 6F hours at JF*FFFrpm. -he D4A mi!rated to a point that as eRuivalent to their density. esults $rom the parental !eneration contained only a sin!le hi!h

density band ; all D4A molecules contained the TheavyT nitro!en.

D4A ta+en $rom the to !enerations a$ter the sitch contained anintermediate;density band ; D4A contained a TheavyT D4A strand $romthe parent and a complementary Tli!htT D4A strand.

Density results $rom !eneration ?* displayed to bands. -hey includedan intermediate density band* composed o$ one parental TheavyT strandand a ne li!ht band* composed o$ only D4A strands ith Tli!htTnitro!en.

Mi3in! the frst and third !enerations shoed all three types o$ D4Amolecules ; heavy* li!ht and intermediate.

4
http://www.uic.edu/classes/bios/bios100/lecturesf04am/meselson_stahl_experiment.htmhttp://www.uic.edu/classes/bios/bios100/lecturesf04am/meselson_stahl_experiment.htmhttp://www.uic.edu/classes/bios/bios100/lecturesf04am/meselson_stahl_experiment.htmhttp://www.uic.edu/classes/bios/bios100/lecturesf04am/meselson_stahl_experiment.htm


22/160

E4 (abelin! o$ D4A'Semiconservative

CsCl ,ra&ient

Ultracentri!',e.

(ensity &i!!erent

E4 (abelin! o$ Proteins


23/160

E4(abelin! o$ Proteins


24/160

Protoco or E4 (a e n! o Prote nHsin! the Sin!le Step 1K2

Competent CellsDay I

E. Inoculate a sin!le colony $rom $reshly strea+ed plate into ml o$ starter culture

6. Gro at ?@oC $or 7L;5 h?. Idem no.E into 6Fml o$ overni!ht cultureJ. Idem no.6 1B E5 h2

Day 6. arvest by centri$u!ation at ?.FFF rpm $or EF m at JoC

L. esuspend cell pellet in 6F ml 1vol eRual to overni!ht culture2o$ induction media' supplemented ith I metal mi3* ithE!/( o$ 4JCl or E4JCl 1Cambrid!e Isotopes2

@. Adapt the cells at ?@oC $or ?F minutes to E h5. Induce ith F.6U 1/v2 rhamnose and F.J mM cAMP overni!ht

at 6oC


25/160

Day ?

. Collect cells by centri$u!ation at .FFF rpm $or6F min at JoC

EF.esuspend cell pellet in 6 ml o$ Cell (ysis

ea!ent ith 6ul o$ 4ase;%ree D4ase andprotease inhibitor coc+tail in F ml

EE. Incubate cell resuspension at roomtemperature $or 6F min

E6.Collect the supernatant by centri$u!ation [email protected] rpm $or 6F min at JoC

E?.(oad ith Protein Purifcation esin* ash o$bindin!/ash bu0er

EJ.,lute ith EF ml o$ elution bu0er


26/160

E4 Incorporation ,:ciencyHnlabeled

1Da2

E4 1Da2 U(abelin

!Monster GreenG%P

6@.?JL [email protected] J.5

C#PII E [email protected] [email protected]?E @.5

C#PII 6 [email protected]@ [email protected] *+e molec'lar $ei,+ts o! 'ri!ie& roteins $ere

&etermine& /y M:1(I *?F mass sectrometry@ CR;PII

roteins $ere reare& an& e8al'ate& !rom t$o searate

cell c'lt'res@ *+eoretical si#e o! 'nla/ele& Monster

Green GFP is AB@D< (a an& o! CR;PII is 4@ (a

molec'lar $ei,+t o! 4=N !'lly s'/stit'te& Monster Green

GFP is AB@B= (a an& CR;PII is 4B@4= (a as


27/160

%i!.


28/160


29/160

E4;labeled C#PII e3pression. Panel A. Proteins $rom uninduced

cells* induced cell lysate supernatant and eluted $rom is(in+ esinere analy=ed by SDS;PAG, on J;6FU -ris Glycine !els $olloed byCoomassie blue stainin!. Panel #. E;E4S)C spectrum o$ 7E mME4 C#PII in EF mM phosphate bu0er ith E?F mM 4aCl. Data asrecorded at the 4ational Ma!netic esonance %acility on a @Fm=

spectrometer eRuipped ith a cryo!enicE

*E

4*E?

C triple;resonanceprobe* data points $or protein and nitro!en.


30/160


31/160

4itro!en (aser' ??@ nm

MA(DI;-


32/160

E* E4* E?C

adioa+tivenuclide

,ner!i 1Me2 al$;li$e1a+tu paruh2

E? 1beta;2

@.65

EJ.F E6.?? y

E4E?4 1betaV2

F.EF6

.?JL EF menit

EEC 1betaV2EJC 1beta;2

EF.LF

?.F6F

6F menit

@?F y

!amma EFF Ke Penetrasi bbrp

cm


33/160


34/160

'etermination by ()* tracer +ineticso, the hal,"li,e o, total body &rotein in

&remature and mature in,ants-

Wut.+e /'* 0eine W* 1lath 2* /rien+e 3. #ased on E4;tracer techniRues important data o$ the

intermediary protein metabolism can be assessed by compartmentanalysis. Xe calculated the hal$;li$e o$ hole body proteins in fve

preterm and fve $ull term in$ants in addition to commonly usedparameters o$ the protein metabolism e.!. protein synthesis rate*protein brea+don rate* 4;turnover rate* si=e o$ metabolic pool*hal$;li$e and reutili=ation o$ aminoacid;4 and the rate o$endo!enous urinary;4. -he in$ants ere a!ed 6@ V/; J and ?E V/;E? days resp. -he hal$;li$e o$ hole body proteins ere $ound to be@. V/; E.5 days in the premature in$ants and thus si!nifcantly

shorter than the EL.F V/; ?.5 days $or the $ull term in$ants. -hedi0erences in the hal$;li$e o$ protein as ell as protein synthesisrate and protein brea+don rate reNect the rapid proteinturnoverin premature in$ants in comparison to $ull term in$ants.

PMID' JFEFL5F WPubMed ; inde3ed $or M,D(I4,Y


35/160

%luorophores/) dot/Stable isotopes


36/160

%luorophores/);dot/Stableisotopes %locytometer/ #io4M/ Mass

SpectrometerInstead o$ usin! optical probes 1Nuorophores

or Ruantum dots2 to identi$y biomar+ers* DS

uses elements or stable isotopes. -here aremore than EFF available elements and stableisotopes that are suitable $or the application.

-he 2yT4FZ uses an application;specifcadaptation o$ an %nducively &oupled 'lasma(ass )pecromeer *%&'-()+to detect andRuanti$y the elemental probes.


37/160

Stable isotope' Xhy It9s4eeded

-he technolo!y addresses an ur!ent need $orimprovement o$ detection andcharacteri=ation o$ cancer cells. -his providesim&roved accuracy and sensitivityo$dia!nosis o$ leu+emia and other diseases*hich are o$ten misdia!nosed or dia!nosedtoo late due to the small number o$ mar+ersused in detection. A hi!h number o$ detecteddisease mar+ersallos $or personali=eddia!nosis and treatment.

154'U2T A113I2ATI4*#6


38/160

154'U2T A113I2ATI4*#62yT4F7

Instrumentation that allosmassively multiple3 analysis o$individual cells or beads'

clinical ' accurate dia!nosis o$disease 1or health2* stem cell

diseases 1e.!.* leu+emia2* ris+;$ree alternative toamniocentesis

research ' cell populationcharacteri=ation* !ene

e3pression* proteininteraction*simultaneous !ene/proteinassay

gene analysis ' multiple3edbeads allo E*FFF[s o$measurements per second
http://www.dvssciences.com/images/cytof_31.jpg


39/160

MAPA

Cy-


40/160

MA81A57 '*AIntercalating /its

-he series o$ MA81A5Z D4A intercalatin! +itsis no available. ,ach +it contains metal;

containin! intercalator* bu0ers* ashes andinstructions. -he E +it is su:cient to label 5FFMillion cells 1e.!.* 5FF sample assays o$ E Millioncells/sample2> the +it is su:cient to label J#illion cells 1e.!.* J*FFF sample assays o$ E

Millions cells/sample2. -he +its are available ithnaturally;abundant Iridium* enriched EEIr orenriched E?Ir. -he enriched intercalators areideal $or discrimination o$ live/dead cells> thenaturally;abundant intercalator is use$ul $or

Ruanti$yin! D4A o$ permeabili=ed or lysed cells.


41/160


42/160

C(s an& intracell'lar


43/160

C(s an& intracell'lar

rotein


44/160


45/160


46/160


47/160


48/160

( # l P h


49/160

(yman* #almer* Paschen*#rac+ett* P$und Series

IC:*. Isotoe Co&e& :!!inity *a,s


50/160

y ,


51/160

i-A) i b i - l ti


52/160

i-A)' isobaric -a! elativeAbsolute )uantitation

-he i-A) ea!ents are the frst set o$multiple3ed* amine;specifc* stable isotoperea!ents that can label all peptides in up toei!ht di0erent biolo!ical samples enablin!simultaneous identifcation and Ruantitation*both relative and absolute* hile retainin!important P-M in$ormation. -here are totypes o$ i-A) ea!ents* $our ple3 and5ple3. -he ne i-A) ea!ents 5ple3 arealso available in more economical bul+ pac+s.


53/160


54/160


55/160


56/160


57/160


58/160


59/160


60/160


61/160


62/160

Isotope-coded afinity tag


63/160

Isotope coded afinity tag(ICAT)

Linker:Heavy versionwill have 13C at *

Light version will have1C at *

Isotope-coded afinity tag


64/160

Isotope coded afinity tag(ICAT)


65/160

!isadvantages o" ICAT

#o Cys resid$es

Cys not accessi%le "or ICAT reagent

Che&ical &odi'cation o" proteins

Tag "rag&entation

eaning o" relative $anti'cationin"or&ation

&elative"'u

ndance

m()


66/160

SI(AC

#table Isotope 3abelin! by AminoAcids in 2ell Culture

#ased on Mass Spectrometry

Simultaneous' ID and Ruanti$ycomple3 protein mi3ture


67/160

-he SI(AC Strate!y

Mammalian cells cannot synthesi=e]essential^ amino acids

I$ labeled analo! o$ AA supplied

instead o$ the natural abundance AA*it ill be incorporated into eachnely synthesi=ed protein chain


68/160


69/160


70/160

EXTRA STEPS


71/160

In Co&parison

Can Ruantitate protein$rom non;livin!

sources* but reRuireschemical modifcationand a:nity steps

Protein populations$rom live cells can be

mi3ed directly a$terlysis and then proteinpurifed.

Xhen you don9t ant tolose protein in e3tra

purifcation steps

hen to $se ICAT hen to $se +ILAC


72/160

In Co&parison

(abels 76FU trypticpeptides 16U relative

abundance o$ cysteine2

Achieves somedecrease in peptidemi3ture comple3ity

(abels more than _tryptic peptides 1EFU

relative abundance o$leucine2

Does not chan!epeptide abundancesresultin! $rom a di!est.

ICAT +ILAC


73/160

,$antitative &assspectro&etry reveals a role

"or the T.ase /ho1p in

actin organi0ation on theperoiso&e &e&%rane

arelli et al2 4

l "


74/160

oals o" paper

15 I&prove &ass spectro&etry toassist in s$%cell$lar locali0ation

!iscri&inate proteins that areco&ponents

o" the organelle "ro&contain&ents

5 Apply this approach to the yeast

peroiso&e


75/160

.eroiso&e

6%i$ito$s organelle

/ids the cell o" toins

eta%olic roles 7-oidation (yeast)

6s$ally sel"-replicate !e novo generation8

htt#*((en+i,i#edia+org(i,i(-mage*.ero/isome+#g


76/160

i8. i8..

Enriched Enriched

+a&ples "or ICATICAT I ICAT II

20g.*

204000 g

#ellet

nriched

for #ero/isomes

and mitochondriaensit$ gradient centrifugation

.e/11#

#"


77/160

+a&ples "or ICATICAT I ICAT II

!iferent yeast strains were $sed 9 is this a pro%l

Yeast strain:BY4743

Yeast straiheterozygodiploid ??

i8. i8..

Enriched Enriched


78/160

ICAT /es$lts

ICAT I ICAT II

p(:) the pro%a%ility o" %eing enriched

hat arethese ICATratios

dependenton8


79/160

ICAT /es$lts

hat are theseICAT ratiosdependent on8

Li&itations o";ass spec5+$%cell$lar peroiso&e enrich&ent

score .ro%a%ility o" %eing enriched in the

enriched peroiso&al &e&%rane"raction as a "$nction o" its ICAT ratio

hy &ight so&e proteins %eenriched in one ICAT %$t notthe other8

?id 4d 4d Bd


81/160

?irst

assess&ent Can cells $se "atty

acids as car%onso$rce8 .eroiso&al 7-

oidation

!elete gene o" interest

row on oleic acid@la$ricacid (L)

Is this a good assay to implicate proteins in peroxi

?id 4d 4d Bd


82/160

?irst

assess&ent Can cells $se "atty

acids as car%onso$rce8 .eroiso&al 7-

oidation

!elete gene o" interest

row on oleic acid@la$ricacid (L)

Is this a good assay to implicate proteins in peroxi

enetic red$ndancy2 nonperoiso&al proteins or other s$%tle

+$%cell$lar distri%$tion o"


83/160

candidates -- !ho"p

!o$%lela%eling$orescence con"ocal&icroscopy

hy hasnDt anyone seen


84/160

y y/ho1p8

l$cose < lycer.las&a

:ndo&e&%ranes

leic acid;.eroiso&es

/ole o" /ho1p in peroiso&e


85/160

p p"$nction

#hy use RHO1-2Ainstead o#$ strain?

rho1-2A:te&p5 sensitiverho1 deletion &$tant

RHO1-2A:rho1-2Awith

rho1 reintrod$ced on aplas&id

rho1-2A cells have"ewer and

s&aller peroiso&es

/ho1p interactions


86/160

/ho1p interactions

GS7&ho1#

GS7&ho1#

GS7&ho1#

".7.e/

".7.e/

".7.e/

Anti%ody against TA.-tag

.eE /ho1 Actin


87/160

.eE-/ho1-Actin

/ho1-.eE arei&portant "ordyna&icasse&%ly@disasse&%ly o" actinon peroiso&es

.ero/isome

Pex25

&ho1

"ctin

T

rho1

pex25


88/160


89/160

4A and 4A #indin!Proteins Participate in ,arly

Sta!es o$ Cell Spreadin!

throu!h Spreadin! InitiationCenters.

Carmen (. de oo!* (eonard `. %oster* and Matthias Mann

Cell May 65* 6FFJ EE@12'LJ;L6

%ocal Adhesions


90/160

%ocal Adhesions

primary sub;cellular macromoleculesthat mediate the re!ulatory e0ects o$e3tracellular matri3 1,CM2 adhesion

on cell behavior 1Chen* 6FF?2. the mechanical lin+a!es to the ,CM*

and as a biochemical si!nallin! hubto concentrate and direct numerous

si!nalin! proteins at sites o$ inte!rinbindin! and clusterin!.

%ocal Adhesions


91/160

%ocal Adhesions

Critical to cellular processes' Motility

Proli$eration

Di0erentiation

e!ulation o$ Gene ,3pression

Survival

Sastry an& ;'rri&,e 2A5556


92/160

MC cells are secondary humanlun! fbroblasts hich under!obeteen LF;@F doublin!s be$ore

senescence.

Goal


93/160

Goal

-o fnd attachment;specifcinteraction partners o$ specifc $ocaladhesion components'

inculin-alin

Pa3illin

MC lun! fbroblast cells

%unctional Analysis o$ theAttachment Proteome by SI(AC


94/160

Attachment Proteome by SI(AC

Strate!y' hi!hli!ht attachment;dependent interactions and not Qustdistin!uish specifc interactions $rom

non;specifc ones.

,3periment


95/160

,3periment

A o'lations o! cells ,ro$nin le'cine-&e!icient me&ia


96/160

Fig.2


97/160


98/160

Electron transitions an& t+eir res'ltin, $a8elen,t+s !or Hy&ro,en


99/160

Electron transitions an& t+eir res'ltin, $a8elen,t+s !or Hy&ro,en@


100/160

Lanthani$e series


101/160


102/160

Hi,+ a!!inity lant+ani&e lin7er


103/160

Isotoe-/ase& Mass Sectrometry


104/160


105/160


106/160

'irect analysis o, dried blood s&ots utili.ingd ti l t
http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1


107/160

desor&tionelectros&rayioni.ation 9'$#I: mass s&ectrometry9;ustin M Wiseman< =>(>

HP1C-ESI MSMS. Meta/olomics
http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1


108/160


109/160

2ell ?iology @ 1roteomics6

I. I. Steart* -he reproducible acRuisition o$ comparativeli id h t h /t d t t d t


110/160

liRuid chromato!raphy/tandem mass spectrometry data$rom comple3 biolo!ical samples. ,apid &ommunicaionsin (ass )pecromery E5 1E2'EL@;E@EF* 6FFJ.

D. . Stover* Di0erential phosphoprofles o$ ,G% and,G% +inase inhibitor;treated human tumor cells andmouse 3eno!ra$ts. &linical 'roeomics E 1E2'L;5F* 6FFJ.

`. A. Caldell* Comprehensive comparative proteomeanalysis. 'roceedings #0h )() &on$erence on (ass

)pecromery and llied opics #;5* 6FF6. #. (arsen* Phosphorylation analysis 1phosmap2 o$

comple3 protein mi3tures. 'roceedings #0h )()&on$erence on (ass )pecrmery and llied opics'EL;ELL* 6FF6.

A. X. #arolet* Administration o$ e3o!enous endothelin;E$olloin! vascular balloon inQury' ,arly and late e0ectson intimal hyperplasia. &ardiovascular ,esearch61?2'JL5;J@L* 6FFE.

IA'adio Immuno Assay


111/160

IA'adio Immuno Assay

Special sa$ety precautions must be observed henper$ormin! IA methods. adioactive isotopes areused by IA tests to label anti!ens or antibodies.Pre!nant $emales should not or+ in an area hereIA tests are bein! per$ormed. Personnel handlin!isotope rea!ents must ear bad!es hich monitor

their e3posure to radiation. Special sin+s and astedisposal containers are reRuired $or disposal o$radioactive aste. -he amount o$ radioisotopediscarded must be documented $or both liRuid andsolid aste. (ea+a!e or spills o$ radioactive rea!entsmust be measured $or radioactivity> the amount o$

radiation and containment and disposal processesmust be documented.


112/160


113/160


114/160


115/160


116/160


117/160


118/160


119/160


120/160

S*EM CE11


121/160

Triple-Enco$in( *ILAC approach


122/160


123/160

;reast Ca SI1:C


124/160


125/160


126/160


127/160


128/160

Silac dru! principle


129/160

! p p


130/160


131/160


132/160

SI1:C samle


133/160


134/160


135/160


136/160

N'cle's

!l'orescence

Mitochondrion


137/160

.ossi'le lin,age 'eteen :/idative " amages

and -nsulin &esistance


138/160

-& $#ergl$caemia

:/idative

Stress

&educed

-nsulin "ction

;icious

C$cle

".

&:S

"'normal gene e/#ression

Somatic Cell Gene utation

o/$guanosine8o/o42?

deo/$guanosine


139/160

!y

All activities o$ the body* incl.-hin+in! (i$tin! a ei!ht idin! a bicycle Maintain a constant body temperature

-o build a stored ener!y supply 1$at2 Hnder restin! 1basal2 conditions* body9s ener!y

is used by'-he s+eletal muscle and the heart 16U2-he brain 1EU2-he +idney 1EFU2-he lever and spleen 16@U2

:*P

,ner!y* heat* or+


140/160

Thermodynamicsis defned as thebranch o$ science that deals ith therelationship beteen heat and other

$orms o$ ener!y* such as or+.

Poer house' eat and A-P


141/160

Harer9 A55


142/160

W+en ;eta

:&renosetor2;eta:R6 ecite9 t+is

sen& si,nal to /ro$n

!atJ $+ic+ is ric+ o!

t+ermo,enin 2Fo

$it+o't F4:*P

synt+ase6 so t+at no:*P $as !orme& /'t

+eat@ Fo $it+o't F4

2t+ermo,enis is also

calle& 'nco'lin,

rotein 2UCP6@ UCPA

$as a/'n&ant inomental !at in o/ese

ale s+aeJ erson

Metabolic/prediabeticsyndrome


143/160

syndrome

Central obese Dyslipidemia

ypertension

Insulinesistance

Atherosclerosis

1CD2

Healt+y

1ittle rone to (ia/etes

It is not eno',+ to /esai& (ia/etes

;eta:R-UCPA-lema7 8isceral-Panas

Source o$ ener!y 1$uel2


144/160

%ood

Krebs cycle Anaerobic

CP/A-P

are use in body $unction?t+er eternal ener,y so'rces9 s'c+ as ra&iant

solar ener,y an& +eat ener,y !rom o'r

s'rro'n&in,s9 can +el mainain /o&y temerat're

/'t are o! no 'sein /o&y !'nction

Ener,y is a /asic concet o! +ysics9 an& is o!

rime imortance in t+e +ysics o! t+e /o&y

E4 in mitochondria


145/160

eal;time study o$ the urea cycle usin! 15Nn.m.r. in

the isolated !!! -he per$used liver as continuously monitored by 15N

n.m.r. spectroscopy at 6F.6@ M= $or !!!mitochondrialcarbamoylphosphate synthetase as rate;limitin!. !!!www.pubmedcenral.nih.gov/ariclerender.$cgi

arid211""080 - +aca sin! meh padhaKasil liyane sa+a .pubmedcentral.nih.!ov CiteH(i+e' MitochondrialMetabolism in Developin!

,mbryos o$ !!! EF 4ov 6FFL !!!-he resultin! Nu3 map shos that

mitochondrialmetabolism in these !!!W15NYalanine*1amino2;W15NY!lutamine* or 1amide2;W15NY!lutamine* !!!www.cieulike.org/.../3... - "0k -Pan!!onan +an!!o nyimpen data sin! is pernah dibu+a+ ; +aca sin! meh padha

Metabolic ,n!ineerin! ' A indo into


146/160

Metabolic ,n!ineerin! ' A indo intocellular metabolism' hepatic !!!

Predicted $ractional abundance o$ thenitro!en mass isotopomers o$ urea as a$unction o$ the $ractional enrichment o$ 15Nin the mitochondrialammonia pool !!!

linkinghub.elsevier.com/rerieve/pii/)1036160"0006"6 - +aca sin! meh padha Kidney International ; Abstract o$ article' -he

intensity o$ !!! Incubation o$ cultures ith W;15NY

!lutamine at p @.J resulted in a !!!derived$rom enhanced Nu3 throu!h both themitochondrialG(D and PDG pathays. !!!www.naure.com/ki/journal/v4#/n4/abs/ki133

41"a hml - +aca sin! meh padha

Spectroscope I


147/160

Mol absorpt a specifc lambda1,\hc/lambda2 H move oneelectron to an orbital ith hi!her ,>

I ma+e hi!her vibration amplitudoatoms hich ti!ht each other1,3cited vibration state2",m'-hermo.

#ase line' EFFU -

ec roma!ne c avespectrum


148/160


149/160

6F.FFF individual particles/s


150/160

-otal


151/160


152/160

Spe+tros+opi nmr 1ba!ianhidro+arbon mole+ul2


153/160

hidro+arbon mole+ul2

Didasar+an pada abs !el radio oleh inti;inti tttdlm mol or!ani+* apabila mol ini berada dlmmedan ma!net +uat

Inti;inti atom unsur;unsur diba!i' a2 mempunyai

spin dan b2 tida+ mempunyai spin Inti berspina+an menimbul+an medan ma!net

+ecil* y! diperi+an oleh suatu momen ma!neti+nu+lir* suatu ve+tor

4u+lida pt! y! berspin inti adl dan C Sedan! E6*L C dan < ta+ punya spin

4u+lida berspin menyerap , ta+ pada radio$re+

yan! sama> pl! la=im dipelaQari d! nmr' proton

11 136

Gri:n 6FFJ


154/160

In the post;!enomic era* several proflin! tools have beendeveloped to provide a more comprehensive picture o$tumour development and pro!ression. -he !lobalanalysis o$ metabolites* such as by mass spectrometryand hi!h;resolution E nuclear ma!netic resonancespectroscopy* can be used to defne the metabolicphenotype o$ cells* tissues or or!anisms. -hese[metabolomic[ approaches are providin! importantin$ormation about tumori!enesis* revealin! netherapeutic tar!ets and ill be an important componento$ automated dia!nosis.

Resonancy


155/160

AWhen the freBuenc$ of the #ulsed radio 'eammatches the freBunc$ of the h$drogen atom4 hich

induces the #rotons to fli#4 this is called the

&esonance @reBuenc$D

A&esonance in & is caused '$ the a'sor#tion of radiation in the

radio freBuenc$4 '$ #rotons in magnetic field


156/160

diabsorbsi inti atom

EJ' Inti atom melalui 8nuclear spin9men!absorbsi!elomban! radio dari$re+uensi tertentu +eti+a ditempat+andalam sebuah medan ma!net +uat

#brp th sblmnya' $re+uensi rensonansi intiatom t!t! ta+ hanya pada E2 +e+uatanmedan ma!net dan 62 tipe atom* tetapiQu!a pada ?2 lin!+un!an +imia atom.

84uclear spins9 dari berba!ai inti dapatsalin! mempen!aruhi* memprodu+sistru+tur;6 halus yaitu seQumlah punca+dalam spe+trum 4M

%agian utama dari s#e,trometer &


157/160

> Ssam#el

Generator fre,uensi radio

ete,tor fre,uensi

radio

Punca+ ttt ut+ atom ttt ELL' Sensitivitas y! tadinya +ecil dapat


158/160

ELL' Sensitivitas y! tadinya +ecil dapatditin!+at+an secara dramati+ Qi+a

pen!!anti berba!ai $re+uensi pende+dipa+ai pulsa intens $re+ radio untu+memberi paparan pada sampel

E@F' per+emban!an cara menentu+an 6

inti berde+atan satu den!an yan! lain 16atom beri+atan/mole+ul2 su+ses pada molrelati$ +ecil tapi !a!al pada y! lebih besar+arena sulit membeda+an resonansi dariinti atom 1spe+trum 4M sepertilapan!an.lan rumput;beribu punca+sh! ta+ mun!+in men!enali punca+ manaut+ atom manaterpecah+an oleh KurtXuthrich

`odoh tiap si!nal 4M den!anproton dalam ma+romole+ul


159/160

\ 8seRuential assi!nment

1985 WFthrich* ara, antara seumlah 'esar inti hidrogen dan mema,ai

informasi ini dengan metode matemati, 'edasar,an geometri ara, untu,

menghitung stru,tur 3 untu, mole,ul+ Saat ini 15720H dari ri'uan

stru,tur #rotein telah ditentu,an dg &+ Ig lain dg ,ristalografi sinar7

J 'e'era a d difra,si ele,tron atau difra,si neutron

Area apli+asi !una 4Mpada ma+romole+ul


160/160

pada ma+romole+ul

Penentuan stru+tur* 4M d! +omplemen +ristalo!raf sinar;

Keun!!ulan 4M bah+an untu+ ba!ian mole+ul tra+terstru+tur dan san!at mobil dalam +ondisi fsiolo!is

Stru+tur protein prion 1mad co' 4obel dlm ilmu +edo+ E@

ut+ Stanley Prusiner2 Stru+tur dan dinami+a ma+romol D4A & 4A

Dlm industri $armasi' ma+romol y! Qadi tar!et mole+ul ba!iobat baru. Mol +ecil obat baru beri+atan d! mol besarmolbesar umumnya berubah. Pentin! ba!i s+rinin! seQumlahcalon obat baru di $ase aal per+emban!an obat baru.

2.isotope application in cell & genetics10.ppt

Documents