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Tissue&Imaging&and&Analysis&for&Drug&Detec6on&using&MALDI&Mass&Spectrometry&Kimberly&A.&Church,&Michelle&K.&Lewis,&Dr.&Krisoffer&Valerie*,&Dr.&Kris6na&Nelson**&
*&Department&of&Department&of&Radia6on&Oncology,&Massey&Cancer&Center,&Goodwin&Research&Laboratory,&Virginia&Commonwealth&University,&Richmond,&VA&&**Chemical&and&Proteomic&Mass&Spectrometry&Core&Facility,&Department&of&Chemistry,&Virginia&Commonwealth&University,&Richmond,&VA&
&Introduc6on& !!
!
!Understanding!the!biochemical!systems!within!a!living!organism!can!be!a!difficult!
task,!though!in!recent!years!matrix:assisted!laser!desorp<on/ioniza<on!(MALDI)!imaging!
mass!spectrometry!(IMS)!has!proven!to!be!an!emerging!method!for!biological!
inves<ga<ons.1!!MALDI!MS!is!oJen!referred!to!as!a!“soJ”!ioniza<on!technique!due!to!its!
capabili<es!in!detec<ng!a!wide!range!of!molecular!weights!(~100!Da!to!>100!kDa)!in!<ssue!
samples.2!!Using!this!method,!a!selected!matrix!is!applied!to!the!specimen!of!interest!and!a!
laser!is!then!used!to!desorb!and!ionize!the!analytes!into!the!gas!phase.2!!For!the!detec<on!
of!pep<des,!proteins,!lipids,!and!drugs,!α:cyano:4:hydroxycinnamic!acid!(CHCA)!is!a!
commonly!chosen!organic!matrix.2,3!!Mass!analyzers!are!then!used!to!determine!the!mass!
to!charge!ra<o!(m/z)!of!the!vola<zed!molecules!and!an!intensity!map!is!created!based!on!
the!individual!ion!peaks!per!each!predetermined!coordinate.4,5!
!In!addi<on!to!proper!matrix!selec<on,!treatments!such!as!<ssue!washing!and!
matrix!recrystalliza<on!are!necessary!to!increase!the!signal!of!a!desired!analyte.!!Direct!
analysis!of!a!<ssue!can!be!limited!due!to!ion!suppression!caused!by!salts!or!other!
compounds!found!within!a!<ssue!such!as!lipids.6!!While!there!are!a!limited!number!of!
sample!prepara<on!protocols!that!address!this!issue,!a!pH!wash!can!be!used!to!remove!
salts!and!suppress!other!compounds!within!in!the!<ssue.6!!Manipula<on!of!the!matrix!aJer!
sublima<on!can!be!achieved!through!recrystalliza<on!and!is!oJen!necessary!to!enhance!
the!signal!of!low!molecular!weight!compounds.7!
Objec6ve&!!
!In!previous!experiments,!KU:60019!was!spo^ed!directly!onto!<ssue!slices!and!
successfully!detected!at!varying!concentra<ons!using!MALDI:IMS.!!A!near:infrared!dye!(IR!
dye!800CW!Carboxylate)!is!normally!used!as!a!biomarker!to!determine!the!localiza<on!of!
the!drug!within!the!brain!aJer!injec<on.!!This!experiment!hoped!to!detect!KU:60019!in!
brain!<ssues!from!mice!that!received!an!injec<on!of!the!drug!and!dye!at!250!μM!and!1!μM!
respec<vely,!and!localize!the!drug!in!the!same!region!as!the!infrared!brain!scans.!!&&
Methods&&Tissue&Prepara6on&!!
!Nude!male!mice!(approximately!8!weeks!old)!were!administered!a!10!μL!dosage!of!
KU:60019!at!250!μM!and!IR!dye!800CW!Carboxylate!at!1!μM!using!CED!(convec<on!
enhanced!delivery)!at!an!injec<on!rate!of!0.5!μL!per!minute.!!The!drug!and!near:infrared!
dye!were!directly!injected!into!the!right!hemisphere!of!the!brain,!leaving!the!leJ!
hemisphere!available!to!be!used!as!a!control.!!Once!the!delivery!of!the!drug!and!dye!was!
complete,!the!mice!were!placed!on!the!Licor!infrared!scanner!and!images!were!collected!
to!visualize!the!near:infrared!sec<on!of!the!brain!containing!the!drug.!!The!brains!were!
then!collected!and!properly!snap!frozen!with!liquid!nitrogen!and!stored!prior!to!sample!
sec<oning.!!!
!Two!brain!sec<ons!were!transversely!cut!at!10!microns!using!a!Leica!CM!1850!UV!
cryostat!and!were!mounted!to!each!slide.!!The!slides!were!then!placed!facedown!onto!the!
Licor!infrared!scanner!and!images!of!the!<ssues!were!collected.!!Figure!1!(A)!includes!an!
image!of!three!mice!brains!containing!the!drug!and!dye!before!sec<oning!while!(B)!shows!
three!slides!with!2!brain!sec<ons!each.!!Addi<onally,!slides!were!spo^ed!in!a!single!area!
with!a!0.25!μL!combina<on!of!the!250!μM!drug!and!1!μM!near:infrared!dye!to!be!used!as!a!
control!spot.!!!
&
Results!!!
!A!preliminary!slide!was!prepared!and!sublimated!with!250!mg!of!CHCA!without!
addi<onal!<ssue!treatments.!!The!drug!was!not!detected!at!548!m/z!and!it!was!determined!that!
addi<onal!<ssue!treatments!would!be!necessary!to!extract!the!analyte!from!the!<ssue!(A).!!The!
drug!was!also!not!detected!in!the!<ssue!when!the!pH!wash!procedure!was!added,!and!the!
control!spot!of!the!drug/dye!combina<on!(250!μM!and!1!μM!respec<vely)!on!the!<ssue!showed!
it!was!not!delocalized!(B).!!!
!An!adapted!recrystalliza<on!method!with!water!was!used!in!conjunc<on!with!the!added!
pH!wash!procedure!and!demonstrated!enhanced!signals!for!the!drug/dye!control!spot.!!While!
the!drug!was!not!detected!at!its!mass!within!the!<ssue!aJer!recrystalliza<on,!the!spo^ed!drug!
showed!stronger!signal!when!compared!to!the!<ssue!from!the!pH!wash!alone.!!The!<ssue!
treated!with!just!the!pH!wash!showed!its!highest!signal!for!the!drug!at!approximately!700.!!AJer!
removing!the!slide!from!the!instrument,!the!second!<ssue!on!the!slide!was!recrystallized!with!
water,!allowed!to!dry,!and!then!reloaded!into!the!instrument.!!Results!for!this!second!<ssue!at!
548.275!m/z!showed!signals!for!the!drug!spot!greater!than!1400.!!!
!!
!
&&&&&
&Conclusion&
!There!are!many!variables!to!consider!when!conduc<ng!<ssue!imaging!using!MALDI:IMS.!!
The!amount!and!distribu<on!of!the!matrix!must!be!op<mized!for!successful!images,!though!high!
matrix!signals!can!interfere!with!the!detec<on!of!low!molecular!weight!analytes.!!Future!
experiments!are!necessary!to!determine!the!proper!<ssue!treatments!necessary!to!detect!a!
signal!for!the!drug!in!the!brain!<ssue!sample.!!When!paired!with!a!near:infrared!dye,!IR!images!
could!be!compared!to!MALDI!images!of!the!drug.!!It!is!also!possible!that!the!bulkiness!of!the!
structure!of!the!dye!in!close!proximity!of!the!drug!is!inhibi<ng!the!signal!of!the!drug!to!be!
detected!within!the!<ssue.!!!
!&
References&!!
1)!!Corne^,!D.;!Reyzer.;!Chaurand,!P.;!Caprioli,!R.!M.!MALDI!image!mass!spectrometry:!molecular!snapshots!of!biochemical!systems.!
!Nature'Methods.!2007,!4,!828:833.!!!2)!!Norris,!J.!L.;!Caprioli,!R.!M.!Analysis!of!Tissue!Specimens!by!Matrix:Assisted!Laser!Desorp<on/Ioniza<on!Imaging!Mass!
!Spectrometry!in!Biological!and!Clinical!Research.!Chemical'Reviews.!2013,!113,!2309:2342.!!!3)!!Goodwin,!R.!J.!Sample!prepara<on!for!mass!spectrometry!imaging:!Small!mistakes!can!lead!to!big!consequences.!Journal'of'
'Proteomics.!2012,!75,!4893:4911.!!!4)!!Svatos,!A.!Mass!spectrometric!imaging!of!small!molecules.!Trends'in'Biotechnol.!2010,!28,!425:434.!!!5)!!Caprioli,!R.!M.!Imaging!mass!spectrometry:!Molecular!microscopy!for!enabling!a!new!age!of!discovery.!Proteomics.!2014,!14,!
!807:809.!
6) Shariatgorji, M.; Källback, P.; Gustavsson, L.; Schintu, N.; Svenningsson, P.; Goodwin, R. J.; Andren, P. E. Controlled-pH Tissue Cleanup Protocol for Signal Enhancement of Small Molecule Drugs Analyzed by MALDI-MS Imaging. Anal. Chem. 2012, 84, 4603-4607.
7) Buck,!A.;!Walch,!A.;!In'situ!drug!and!metabolite!analysis!in!biological!and!clinical!research!by!MALDI!MS!imaging.!Bioanalysis.!!2014,&6,!1241:1253.!!
8) h^p://www.licor.com/bio/products/reagents/irdye/800cw/structure.html!(Accessed!May!1,!2015).!!
9) Golding,!S.!E.;!Rosenberg,!E.;!Valerie,!N.;!Hussaini,!I.;!Frigerio,!M.;!CockcroJ,!X.!F.;!Chong,!W.!Y.;!Hummersone,!M.;!Rigoreau,!L.;!
!Menear,!K.!A.;!O'Connor,!M.!J.;!Povirk,!L.!F.;!van!Meter,!T.;!Valerie,!K.!Improved!ATM!kinase!inhibitor!KU:60019!radiosensi<zes!
!glioma!cells,!compromises!insulin,!AKT!and!ERK!prosurvival!signaling,!and!inhibits!migra<on!and!invasion.!Molecular'Cancer''TherapeuGcs.'2009,'8,!2894:2902.&
!!
&Acknowledgements!!
I!would!like!to!thank!the!following!people!for!making!this!project!possible:!Dr.!Valerie!and!Dr.!Nelson!
for!their!guidance!and!exper<se,!Michelle!Lewis!for!her!<me!and!help!conduc<ng!experimental!
trials,!Kevin!Kni^er!for!his!construc<on!of!the!vacuum!pump!set!up,!and!Mary!Tokarz!for!providing!
guidance!with!<ssue!sec<oning.!!!
!!
&
Tissue&Washing&!!
!In!order!to!op<mize!the!signal!of!the!drug!within!the!<ssue,!the!Shariatgorji!et'al.!buffered!pH!wash!protocol!was!adopted!for!this!experiment.6!!First,!a!100!mM!solu<on!(pH!6)!of!ammonium!acetate!
was!added!to!a!60!mL!slide!holder.!!The!slide!was!then!submerged!into!the!solu<on!for!10!seconds!and!
promptly!removed.!!The!slide!was!allowed!to!dry!for!approximately!20:25!minutes!before!proceeding!to!
the!matrix!applica<on!procedure.!!!!
!
Matrix&Sublima6on&&
!Once!the!apparatus!was!in!place!on!the!hot!plate,!the!vacuum!pump!(Alcatel!model)!was!
connected!to!the!chamber!and!the!system!was!run!at!reduced!pressure!for!15!minutes!with!vacuum!valve!
slightly!open.!!Figure!2!(A)!shows!the!set!up!of!the!sublima<on!chamber!on!the!hot!plate!with!the!vacuum!
pump!connected.!!Cold!water!(~15!°C)!was!added!to!the!water!chamber!for!an!addi<onal!5!minutes.!!The!
hot!plate!was!then!turned!to!the!5½&marker!(~100!°C)!and!the!vacuum!valve!remained!open!for!the!
dura<on!of!the!sublima<on!process.!!The!first!signs!of!sublima<on!occurred!around!7!minutes!and!by!9!
minutes!there!was!a!dis<nct!ring!of!vapors!that!appeared!on!the!glass!above!the!foil!cup!but!below!the!
slide.!!At!ten!minutes,!a!fair!amount!of!the!matrix!had!sublimated,!crea<ng!a!greenish:yellow!ring!below!
the!water!chamber.!!At!approximately!twelve!minutes!the!heat!was!turned!off,!the!vacuum!valve!was!
closed,!and!vacuum!pump!was!turned!off.!!The!apparatus!was!allowed!to!cool!to!room!temperature!for!
5:7!minutes!before!unclamping!the!pieces!and!removing!the!cold:water!chamber!with!the!slide!a^ached.!
!!
!!
Figure!1.!!Three!brains!were!scanned!aJer!they!were!snap!frozen!with!liquid!nitrogen.!!The!green!fluorescence!is!the!dye!
corresponding!to!the!loca<on!of!the!drug!(A).!!Three!slides!were!prepared!using!sec<oned!por<ons!from!a!single!brain!
(B).!!The!slide!on!the!leJ!was!an!uncoated!slide!while!the!right!two!slides!are!ITO!coated!slides.!!(C)!is!the!chemical!
structure!of!IR!dye!800CW!Carboxylate!(C46H50N2Na4O15S4)!which!a!molecular!weight!of!1091.11!g/mol.8!!!
(A)!
KUX60019!C30H33N3O5S!
MW:!547.67!g/mol!
Figure!3.!KU:60019!is!an!ATM!kinase!
inhibitor!used!to!radiosensi<ze!human!
glioma!cells.9!!The!structure!is!pictured!
above,!including!formula!and!molecular!
weight!(A).9!!A!mass!spectrum!was!also!
obtained!for!KU:60019,!indica<ng!a!mass!to!
charge!ra<o!of!548.192!m/z!(B).!!
(B)!
(A)! (B)!
Figure!2.!The!vacuum!pump!is!connected!to!the!sublima<on!chamber!(A)!and!aJer!~10!
minutes!with!heat,!the!sublima<on!of!CHCA!is!visible!inside!the!chamber!(B).!!Variable!
pressure!from!the!vacuum!pump!resulted!a!gradient!of!matrix!coverage!results!(C).!!
Once!sublima<on!was!complete,!the!slide!was!taped!inside!the!top!of!a!Petri!dish!to!
perform!the!recrystalliza<on!procedure!(D).!!!
(A)! (B)! (C)!
Figure!4.!MALDI:IMS!did!not!detect!the!drug!at!its!mass!for!issues!with!an!injec<on!of!the!drug!and!dye!(A).!!Addi<onal!
<ssues!treated!with!the!pH!wash!showed!a!control!spot!of!the!drug/dye!was!not!delocalized!and!the!background!noise!was!
reduced!(B).!!Recrystalliza<on!with!water!doubled!the!signal!of!the!control!spot,!but!did!not!detect!the!analyte!within!the!
<ssue!(C).!!!
(A)!
(B)!
O
NN
OS
O
O
N
O
!!
!!
(D)!
Recrystalliza6on&!!
!A!recrystalliza<on!method!was!developed!based!on!the!protocol!described!by!Zimmerman!et'al.7!!!
A!beaker!of!deionized!water!was!brought!to!a!boil!on!hot!plate!and!the!sublimated!slide!was!taped!to!a!
stainless!steal!plate!a^ached!to!the!underside!of!the!top!part!of!a!Petri!dish!using!copper!thermal!
conduc<ve!tape.!!The!plate!was!turned!upside:down!and!held!3!cm!above!the!boiling!water!for!60!
seconds,!collec<ng!condensa<on!on!the!slide.!!The!slide!was!then!allowed!to!completely!dry.!!This!
procedure!was!also!conducted!three!<mes!on!one!slide,!wai<ng!90!seconds!between!the!60!second!
intervals!where!the!slide!is!exposed!to!water!vapor.!!!
&
SO2Na
O
N
SO2Na
SO2Na
N
CO2Na
SO2-
(C)!
(C)!
