2.isotope application in cell & genetics10.ppt
TRANSCRIPT
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
1/160
Isotope application in Cell
& Genetics
Peni KS Mutalib
Medical Physics Department
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
2/160
Content
Defnition/ Abbreviation
Sample/ ea!ent"Cell culture
#asic Principle o$ Instrument Methods
%luophoreSemiconductorStable
isotope So$tare' ICP( )uant* Cytocluse*
Cyto)uic+* MultiS,-* Cell)uest* etc.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
3/160
Same * di0erent A' IsotopicSame A* di0erent ' Isobaric
Isotope is a same chemical elementith'
Same number o$ 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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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! $or elative and Absolute )uantitationh,SC' human ,mbryonic Stem Cell
(C/MS ;; GC/MS ;;MS/MS"%lo cytometer/MSMA(DI;-
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
5/160
Abbr ITUband Frequency Wavelength
B ? = EFF*FFF +m
,(% E ??F = EFF*FFF +m EF*FFF +m
S(% 6 ?F?FF = EF*FFF +m EFFF +mH(% ? ?FF?FFF = EFFF +m EFF +m
(% J ??F += EFF +m EF +m
(% ?F?FF += EF +m E +m
M% L ?FF?FFF += E +m EFF m
% @ ??F M= EFF m EF m
% 5 ?F?FF M= EF m E m
H% ?FF?FFF M= E m EFF mm
S% EF ??F G= EFF mm EF mm
,% EE ?F?FF G= EF mm E mm
Above ?FF G= B E mm
HP-MW-FIR-IR-NIR-VIS-UV-X-Ray-Electron-Positron-Gamma Ray Raman
Micromol-nanomol-icomol-!emtomol"atto-#eto-yocto
NMR-MRI"MS"Flo$ 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 -
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
6/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
7/160
Fluorophores: PEFITC--PerCP-Cy5.5APC: Phyco Erythrin- Fluorescein
Isothiocyanate-Allophycocyanine%-&ot semicon&'ctors
CD45: LeucocyteCD3: T Lyphocyte
CD4: Th
CD!: T"
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
9/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
10/160
J55 nm EL;L nm
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
11/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
12/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
14/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
15/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 $it+ little or no si,nal &istortion9
res'lts in +i,+ recision an& t+ro',+'t
a/sol'te 0'antitation
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
19/160
+4,*+45$
+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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 -
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 -
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
22/160
E4 (abelin! o$ D4A'Semiconservative
CsCl ,ra&ient
Ultracentri!',e.
(ensity &i!!erent
E4 (abelin! o$ Proteins
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
23/160
E4(abelin! o$ Proteins
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 $or ?F minutes to E h5. Induce ith F.6U 1/v2 rhamnose and F.J mM cAMP overni!ht
at 6oC
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
25/160
Day ?
. Collect cells by centri$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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
27/160
%i!.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
28/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 $or protein and nitro!en.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
30/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
31/160
4itro!en (aser' ??@ nm
MA(DI;-
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
32/160
E* E4* E?C
adioa+tivenuclide
,ner!i 1Me2 al$;li$e1a+tu paruh2
E? 1beta;2
@.65
EJ.F E6.?? y
E4E?4 1betaV2
F.EF6
.?JL EF menit
EEC 1betaV2EJC 1beta;2
EF.LF
?.F6F
6F menit
@?F y
!amma EFF Ke Penetrasi bbrp
cm
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
33/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
34/160
'etermination by ()* tracer +ineticso, the hal,"li,e o, total body &rotein in
&remature and mature in,ants-
Wut.+e /'* 0eine W* 1lath 2* /rien+e 3. #ased on E4;tracer techniRues important data o$ 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 $or M,D(I4,Y
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
35/160
%luorophores/) dot/Stable isotopes
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
36/160
%luorophores/);dot/Stableisotopes %locytometer/ #io4M/ Mass
SpectrometerInstead o$ 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.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 $or personali=eddia!nosis and treatment.
154'U2T A113I2ATI4*#6
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
38/160
154'U2T A113I2ATI4*#62yT4F7
Instrumentation that allosmassively multiple3 analysis o$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$measurements per second
http://www.dvssciences.com/images/cytof_31.jpg -
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
39/160
MAPA
Cy-
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
40/160
MA81A57 '*AIntercalating /its
-he series o$ 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.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
41/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
42/160
C(s an& intracell'lar
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
43/160
C(s an& intracell'lar
rotein
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
44/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
45/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
46/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
47/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
48/160
( # l P h
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
49/160
(yman* #almer* Paschen*#rac+ett* P$und Series
IC:*. Isotoe Co&e& :!!inity *a,s
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
50/160
y ,
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
51/160
i-A) i b i - l ti
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
52/160
i-A)' isobaric -a! elativeAbsolute )uantitation
-he i-A) ea!ents are the frst set o$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* $our ple3 and5ple3. -he ne i-A) ea!ents 5ple3 arealso available in more economical bul+ pac+s.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
53/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
54/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
55/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
56/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
57/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
58/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
59/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
60/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
61/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
62/160
Isotope-coded afinity tag
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
63/160
Isotope coded afinity tag(ICAT)
Linker:Heavy versionwill have 13C at *
Light version will have1C at *
Isotope-coded afinity tag
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
64/160
Isotope coded afinity tag(ICAT)
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
65/160
!isadvantages o" ICAT
#o Cys resid$es
Cys not accessi%le "or ICAT reagent
Che&ical &odi'cation o" proteins
Tag "rag&entation
eaning o" relative $anti'cationin"or&ation
&elative"'u
ndance
m()
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
66/160
SI(AC
#table Isotope 3abelin! by AminoAcids in 2ell Culture
#ased on Mass Spectrometry
Simultaneous' ID and Ruanti$ycomple3 protein mi3ture
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
68/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
69/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
70/160
EXTRA STEPS
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 $se +ILAC
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
72/160
In Co&parison
(abels 76FU trypticpeptides 16U relative
abundance o$ cysteine2
Achieves somedecrease in peptidemi3ture comple3ity
(abels more than _tryptic peptides 1EFU
relative abundance o$leucine2
Does not chan!epeptide abundancesresultin! $rom a di!est.
ICAT +ILAC
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
73/160
,$antitative &assspectro&etry reveals a role
"or the T.ase /ho1p in
actin organi0ation on theperoiso&e &e&%rane
arelli et al2 4
l "
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
75/160
.eroiso&e
6%i$ito$s organelle
/ids the cell o" toins
eta%olic roles 7-oidation (yeast)
6s$ally sel"-replicate !e novo generation8
htt#*((en+i,i#edia+org(i,i(-mage*.ero/isome+#g
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
76/160
i8. i8..
Enriched Enriched
+a&ples "or ICATICAT I ICAT II
20g.*
204000 g
#ellet
nriched
for #ero/isomes
and mitochondriaensit$ gradient centrifugation
.e/11#
#"
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
77/160
+a&ples "or ICATICAT I ICAT II
!iferent yeast strains were $sed 9 is this a pro%l
Yeast strain:BY4743
Yeast straiheterozygodiploid ??
i8. i8..
Enriched Enriched
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
78/160
ICAT /es$lts
ICAT I ICAT II
p(:) the pro%a%ility o" %eing enriched
hat arethese ICATratios
dependenton8
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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"
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
83/160
candidates -- !ho"p
!o$%lela%eling$orescence con"ocal&icroscopy
hy hasnDt anyone seen
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
84/160
y y/ho1p8
l$cose < lycer.las&a
:ndo&e&%ranes
leic acid;.eroiso&es
/ole o" /ho1p in peroiso&e
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
85/160
p p"$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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
86/160
/ho1p interactions
GS7&ho1#
GS7&ho1#
GS7&ho1#
".7.e/
".7.e/
".7.e/
Anti%ody against TA.-tag
.eE /ho1 Actin
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
88/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
91/160
%ocal Adhesions
Critical to cellular processes' Motility
Proli$eration
Di0erentiation
e!ulation o$ Gene ,3pression
Survival
Sastry an& ;'rri&,e 2A5556
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
92/160
MC cells are secondary humanlun! fbroblasts hich under!obeteen LF;@F doublin!s be$ore
senescence.
Goal
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
93/160
Goal
-o fnd attachment;specifcinteraction partners o$ specifc $ocaladhesion components'
inculin-alin
Pa3illin
MC lun! fbroblast cells
%unctional Analysis o$ theAttachment Proteome by SI(AC
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
94/160
Attachment Proteome by SI(AC
Strate!y' hi!hli!ht attachment;dependent interactions and not Qustdistin!uish specifc interactions $rom
non;specifc ones.
,3periment
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
95/160
,3periment
A o'lations o! cells ,ro$nin le'cine-&e!icient me&ia
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
96/160
Fig.2
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
97/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
98/160
Electron transitions an& t+eir res'ltin, $a8elen,t+s !or Hy&ro,en
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
99/160
Electron transitions an& t+eir res'ltin, $a8elen,t+s !or Hy&ro,en@
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
100/160
Lanthani$e series
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
101/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
102/160
Hi,+ a!!inity lant+ani&e lin7er
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
103/160
Isotoe-/ase& Mass Sectrometry
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
104/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
105/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 -
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
107/160
desor&tionelectros&rayioni.ation 9'$#I: mass s&ectrometry9;ustin M Wiseman< =>(>
HP1C-ESI MSMS. Meta/olomics
http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1http://xlink.rsc.org/?DOI=b922329k&RSS=1 -
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
108/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
109/160
2ell ?iology @ 1roteomics6
I. I. Steart* -he reproducible acRuisition o$ comparativeli id h t h /t d t t d t
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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$olloin! vascular balloon inQury' ,arly and late e0ectson intimal hyperplasia. &ardiovascular ,esearch61?2'JL5;J@L* 6FFE.
IA'adio Immuno Assay
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
111/160
IA'adio Immuno Assay
Special sa$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.
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
112/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
113/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
114/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
115/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
116/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
117/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
118/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
119/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
120/160
S*EM CE11
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
121/160
Triple-Enco$in( *ILAC approach
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
122/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
123/160
;reast Ca SI1:C
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
124/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
125/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
126/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
127/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
128/160
Silac dru! principle
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
129/160
! p p
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
130/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
131/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
132/160
SI1:C samle
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
133/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
134/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
135/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
136/160
N'cle's
!l'orescence
Mitochondrion
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
137/160
.ossi'le lin,age 'eteen :/idative " amages
and -nsulin &esistance
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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/$guanosine
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
139/160
!y
All activities o$ 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+
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
141/160
Harer9 A55
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
$as a/'n&ant inomental !at in o/ese
ale s+aeJ erson
Metabolic/prediabeticsyndrome
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
143/160
syndrome
Central obese Dyslipidemia
ypertension
Insulinesistance
Atherosclerosis
1CD2
Healt+y
1ittle rone to (ia/etes
It is not eno',+ to /esai& (ia/etes
;eta:R-UCPA-lema7 8isceral-Panas
Source o$ ener!y 1$uel2
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
144/160
%ood
Krebs cycle Anaerobic
CP/A-P
are use in body $unction?t+er eternal ener,y so'rces9 s'c+ as ra&iant
solar ener,y an& +eat ener,y !rom o'r
s'rro'n&in,s9 can +el mainain /o&y temerat're
/'t are o! no 'sein /o&y !'nction
Ener,y is a /asic concet o! +ysics9 an& is o!
rime imortance in t+e +ysics o! t+e /o&y
E4 in mitochondria
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
145/160
eal;time study o$ 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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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$rom enhanced Nu3 throu!h both themitochondrialG(D and PDG pathays. !!!www.naure.com/ki/journal/v4#/n4/abs/ki133
41"a hml - +aca sin! meh padha
Spectroscope I
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
147/160
Mol absorpt a specifc lambda1,\hc/lambda2 H move oneelectron to an orbital ith hi!her ,>
I ma+e hi!her vibration amplitudoatoms hich ti!ht each other1,3cited vibration state2",m'-hermo.
#ase line' EFFU -
ec roma!ne c avespectrum
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
148/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
149/160
6F.FFF individual particles/s
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
150/160
-otal
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
151/160
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
152/160
Spe+tros+opi nmr 1ba!ianhidro+arbon mole+ul2
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
153/160
hidro+arbon mole+ul2
Didasar+an pada abs !el radio oleh inti;inti tttdlm mol or!ani+* apabila mol ini berada dlmmedan ma!net +uat
Inti;inti atom unsur;unsur diba!i' a2 mempunyai
spin dan b2 tida+ mempunyai spin Inti berspina+an menimbul+an medan ma!net
+ecil* y! diperi+an oleh suatu momen ma!neti+nu+lir* suatu ve+tor
4u+lida pt! y! berspin inti adl dan C Sedan! E6*L C dan < ta+ punya spin
4u+lida berspin menyerap , ta+ pada radio$re+
yan! sama> pl! la=im dipelaQari d! nmr' proton
11 136
Gri:n 6FFJ
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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 &
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
157/160
> Ssam#el
Generator fre,uensi radio
ete,tor fre,uensi
radio
Punca+ ttt ut+ atom ttt ELL' Sensitivitas y! tadinya +ecil dapat
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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
-
7/24/2019 2.Isotope application in Cell & Genetics10.ppt
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.