Methodologies for Metabolomics

EXPERIMENTAL STRATEGIES AND TECHNIQUES

Edited by

Norbert W. LutzUniversity of Aix-Marseille, France

Jonathan V. SweedlerUniversity of Illinois, Urbana-Champaign, USA

Ron A. WeyersUniversity Medical Centre Nijmegen, the Netherlands

UCAMBRIDGE:': UNIVERSITY PRESS

CAMBRIDGEUNIVERSITY PRESS

University Printing House, Cambridge CB2 885, United Kingdom

Published in the United States of America by Cambridge University Press, New York

Cambridge University Press is part of the University of Cambridge.

It furthers the University's mission by disseminating knowledge in the pursuit ofeducation, learning and research at the highest international levels of excellence.

www.cambridge.orgInformation on this title: www.cambridge.org/9780521765909

© Cambridge University Press 2013

This publication is in copyright. Subject to statutory exceptionand to the provisions of relevant collective licensing agreements,no reproduction of any part may take place without the writtenpermission of Cambridge University Press.

First published 2013

Acatalogue record for this pubffcation is available from the British Library

Library ofCongress Catafoguing in Publication data

Methodologies for metabolomics: experimental strategies and techniques /[edited by] Norbert W.lutz, Jonathan V. Sweedler, Ron A. Weyers.

p.; cm.Includes bibliographical references and index.ISBN 978-0-521-76590-9 (hardback)I. lutz, Norbertw', 1952- II. Sweedler, Jonathan V. 111. Weyers, Ron A., 1951­[DNlM: 1. Metabolomics - methods. 2. Mass Spectrometry - methods.3. Metabolic Diseases - diagnosis. 4. Metabolome. QU 120]543'.65-dc23 2012013907

ISBN 978-0-521-76590-9 Hardback

Cambridge University Press has no responsibility for the persistence or accuracy ofURLs for external or third-party internet websites referred to in this publication,and does not guarantee that any content on such websites is, or will remain, accurateor appropriate.

• Direct Metabolomics from Tissues and Cells:Laser Ablation Electrospray Ionization forSmall Molecule and Lipid CharacterizationAkos Vertes, Bindesh Shrestha, and Peter Nemes

140

The y tematic study of metabolite. and th ir rdat d pathway in variou organi '01

i xperiencing a renai ance. In the fir t half f the twentieth century studyingelect metabolic proce e played a ntral role in biochemi try and brought about

maj r di coveri . uch a th rnithin and the citric acid cycle (Kornberg, 20 0).From the middle of the century, incr a ingl pow rful tool u ed in metab Ii 01

re arch in luded efficient separation method!> (ga chromatography fa ] and high­pcrformance liquid chr matograph ) combined with pectrometer. aim d at . mallmolecule. (nuclear magnetic re onanc I MR] and rna p ctr metry [M ]).10 the1970. and early 19 Os. genetic took ccntcr stagc, aided by the introduction of D A,equencing, polymerase chain reaction (P R), ami rccombinant DNA techn logie .

In the late 1980s, with the introducti n of oft i nization methods in M ,the y.­tematie investigation of peptide and pI' tein' became po sible, and proteomi waborn. During the 1990 and the iiI' t decade of the twenty-fir t century, re earcher ingenomic accompli h d the equencing f genome in multiple species. The devel-

pm nL of microarray techniqu gave u!> powerful tool t tudy the tran. criptome.and, with the help of n w technique' in M . inve tigator in proteomic began thechallenging ta 'k of mapping the corre ponding pI' team .

M tabolomics - the y tematic inve tigation of the metabolite and metab licprocee' in an organism - i, lagging behind wing to multiple chall nge . In con­tra't t nucleic acid and protein', metabolite are tructuralJ diver e. A: a on­sequenc ,their eparation and spectra.corie identification require a wide varietof protocol, Metabolite concentration al vary ext n ively in pace and time; (orexample. the temporal chang are more rapid than in the ca e of biopolym r', Typi­cal change' in the transcriptome can take minute, . hours, or longer (Ye et aI., 2009),wh rca protein and metabolite conc ntration. vary in the range of minute' andeconds. onventional metabol mic protocol. rely n rapid sampling that includes

quenching of the biochemical proce e and often derivatization before separalionand :pectrometric analy i ( mart et aI., 2010). Becau f the'e complex tep. it ialway que tionable how cl to it native tat the tudied y ·tem has remained.

To explore the native m tabolom , n w meth d are needed that reo pond tovari u mall organic molecule on a timecale of econd . Ideally, the e t chnique'have to be capable f local anal i and exhibit ufficiently high en iti ity. llecaumetabolite molecule are much mailer than nucleic acid I' protein, th yare more

1. Direct Tissue Analysis and Imaging

prone to patial redi tribution during ample preparation. The local analy is andmol cular imaging of metabolite in tis ue and cell al 0 require. pecial con id­erations (ee hapter. 18, 21, and 22 (or noninvasive and nondcstructiv in vivom tabolic imaging by MR technique ).

The advent o( atmo 'phcric pre 'ur i nization methods ( I' MS ha enabled thedirect analy i of ti. ue and cell with little or no ample preparation. Thi rapidlxpanding group of technologie ha: be n u d xten i ely t analyze biologicalample dir ctl and include ambi nt d orption lectro pray ionization (D I)

(Takats et aI., 2004), direct analysi- in realtime (DART) ( ody ct aI., 2005), extrac­ti e electro pray ionization ( ESI) ( hen et a!.. 2006a, 2007), atmospheric pre ­ur infrared matrix-a si ted la er de rpti n ionization (AP fR-MALDI) (Li et aI.,

_ 7' Verte et al.. 200), lectro pray-a' i ted la er de 'orption ionization ( LDI)hiea et aI., 2005), and 1a er ablation electro pray ionization (L I) ( ernc and

\ rte, 2007). Man of th 'e techniqu can be charact rized a in itu or. in mee , in vivo method ( erne and Verte., 2012). Although the analy i acrific.

me cell of lh ludied organi 'm, it i Ie obtrusive than biop 'ie . Ultimat Iy,. am­pIing can be arranged so that a living organi m can be vieweLi unperturbed, a long

the ampl d volume i igniiieantly 'maller than the volumc of the organ! m. Thi.hapter focu e. n the application. o( LAE TM for the dir ct analy i of ti ue

and cell with t\: o-dimen ional and thre -dimen ional imaging and depth profllingf mall metab lile and lipid.

1. Direct Tissue Analysis and Imaging with Laser AblationElectrospray Ionization

In the field of biom dical analy. i., L .. I M ha exhibit d con iderable uccesm interrogating thc -mall m lecule comp ilion of biological fluid uch a' bl d

La rna and urine a well a plant, animal. and human ti uc and ingle cell. Therear numerou fundamental and unique a 'P ct that place LA I M among the

nabling mcthoLiologi s in direct m tabolomic inve ligaLi n .In LAE 1 M ,the sampling tep is 'palially ind pcndent from that of i n gen­

ralion. Fir t, the native water molecul . of the. amplc re excited by a [ocu dmid-infrared la 'cr light. Al a wa elength of 2,940 nO', rapid energy depo 'ilion

cur through the e citation of the vihralional moll of water molecule. Pro­id d that uffici nt energy i c upled into the ampl ,th re ult of th pr c '

pha e cxplo. ion-dri en ablation that xp I particular mattcr within a fe hun­dr d micr ec nd (Apitz and Vogel, 2005; hen et aI., 200 b; h n and Vert ,

), and the momentum gain d by the ejectiles tran. ports lhem to everallen. ofmillimeter above the sample ur[ac ( em and Vertc ,2007).

n electro pray 'ource, pI' ferably p rated in the c n -jct mode, efficientlyenerate a cloud of mall charged dr pI t ( me' et al. 2007) that int rcept thearticle ablated (r m the ample (em and Vert .2007). the droplel coa­

ce with the particulate matter, the metaholite of the sample are transferred into abarged olution-pha cd microenvironment. Ultimately, metabolite are converted

oft ion via pr c s s similar to ion production during conventional electro:praylOnization ( 1) (Nemes et aI., 2012). orrcspondingly. wilh LAB I MS, am piescan be analyzed wilhin second, pr viding a nap hot f th (a t- volving m tabolit

141

142 Direct MetaboJomics from Tissues and Cells

compo ition. Tbi f rm the ba i f high-throughput and large- 'cal inve tigationu ing LAE I MS.

1.1. Label-Free Metabolite Identification

The ion generated in LAE I can be analyzed by any rna pectr meter that iequipped with an atmo pheric pre 'ure interface. Mo·t commonly a commercial

I or atmo pheric pre ure chemical ionizati n source i replaced by a cu tom­built LAE I ion ource. More recently, the introducti n of commercial LAE Iion ource' ha been announced (Protea Bio ciences, Inc., Morgantown, WV). TheLAE I sy tern has been ucce fully implement d on time-of-flight and ion trapin truments that feature quadrupole and hexapole ion guide for enhanced iontran fer and collisional activation. The ma spectrometer can be operated in ingle-tage or tandem mode to facilitate chemical identification (Neme and Vertes,

2007).The application of LAE I MS in metabolomic follows different workflows for

metabolite di covery than for the tructural identification of known compounds.The latter typically employ a multistep M -ba ed approach. Accurate mas -to­charge ratio (m/z) mea urement i the starting point in the tructure identification byLAE 1M. Ions are mea ured with m/z accuracies uch a Ie than 5 ppm, althoughfor targ ted compounds lower accuracies can al 0 yield atisfactory re ult . Themea ured mlz value are compared against metabolomic database for prokaryotic.plant (e.g., http://www.plantcyc.orgl), and animal speci a well a. human ample(e.g., http://www.hmdb.ca/). There i a growing number of multiorganism pathwaydatabase integrated with g nomic and prot omic data (e.g. http://metacyc.org/,http://www.genome.jp/kegglpathway.html) that can al 0 help ideotit1cation.

I"otope llistribution patterns can also be determined to narr w the Ii t of puta­tive compound further. The remaining po itive matche are evaluated in tandemM experiment whereby the fragmentation behavior f the unknown ion i com­pared with that of chemical tandard. In certain ca s, a reactant can be added tothe lectro prayed solution to tran form th analyte chemically. Here the objectivei to improve the ion yield or facilitate structure-specific fragmentation. The lattercan be beneficial for lipid that are known to yield a very limited number of frag­ment in collision-activated di. ociation from protonated precursors. Introducinglithium ion via the electrosprayed olution leads to the generation of lithiated lipidspecie that readily fragment into numerous characteristic product ion that help tdecipher the structure of the parent lipid molecule (Shre tha et a1.. 2010a). Thi mul­ti tep elucidation chern for known metabolite u ually yields identifications withhigh analytical confidence far biomedical ample.

In th metabolite di covery mode (i.e.. in case where a particular metabolitehas not been described in the literature for a given peci s), the u e of databa ei of limited help. In the e ituation., tandem M -ba ed technique' are manda­tory, and orthogonal analytical technique are needed to enhance the confidence ofidentification further. The latter include offline experiments u ing cla' ic eparatianmethod (e.g., liquid chromatography with M detection). An appealing yet unu edalternative i coupling LAESI with ion mobility pectrom try (Bohrer et aI., 2008),which could eparate tructural i omers. In thi xperim nt, the time of LA lin

1. Direct Tissue Analysis and Imaging 143

Figurc 6.1. chematic of the LAElMapparatus for metabolic inve tigationdirectly from tissue.. The ample ( ),po iti ned on a amp] holder (SH), Lablatcd by a mid-infrared la.er focu edby a f cu ing len (FL), and the abla­ti n plumc (red dots) i intercepted bycharged droplet (green dots) generatcdb an lectro pray ource (ES). Thechargcd droplet are eeded with theneutral ablated material. which rc ulltn the production of ample- pecific ionthat are analyzed by a ma pectrom­etcr (M ). The ample is optionallycooled or kept frozen by a Peltier stage(PT) equipped with a heat 'ink (HS)and fans.

eneration would be synchronized with the injection of ion into the ion mohil­it cell. Combining LAE I with ion mobility spectrometry rai es the prospect ofimproving confid nce in metabolite identification while maintaining fa t amplanaly i .

1.2. Metabolic Analysis

Th flexibility that re ult from decoupling ampling from ion generation ha far-hing benefit. in practical m tabolomic investigation. Perhaps one of the most

auractive feature of LAESI M i that the biomedical analy i is facilitated byb inherent water content of th ample. A hown in Figure 6.1, LAESI M11 w direct metabolic analy i becau e it eliminate the requir ment to modify

biological or m dical ample. For example, certain metabolite and xenobioticI cule have been analyzed in water-containing ample. uch a bodily fluids,

lthin econd without any ample preparation. xampl how that small metabo-(e.g., carnitine, phosphocholin , tetradecenoylcarnitine) and lipid molecules

- 0" glycerophosphocholine [P ]; the lipid nomenclature accepted by the LIPIDPeon ortium [http://www.lipidmaps.org/] i used throughout Ihi' chapter) can

r adily detected, and th excr tion of drug molecules (e.g., Ihe antihistaminef nadine) can be monitored directly from whole blood and urine (Neme and

n ,2007). LAE I ma spectra often include multiply charged ion, a phe­enon typically obs rved in E 1. Deconvolution of Ih ion charge tate revealdant protein in human blood, including the serum albumin and the Ci andain of hemoglobin. Thus, the molecular ma range of LAE I MS analy io from mall metabolites to macromolecules greater than 66 kDa.L al analy is i another important feature of the LAE I M method. The anal­

ea of the laser beam j typicalJy approximately 200 ~m in diameter and canr duc d to 50 ~m without ignificant ignal 10 . The ablation dimen ion can

fully adju ted for various tis ue and cell type to upp rt in itu. ex vivo,~ vi 0 experiment. The laller can be ob erved in Figur 6.2, which how the

144 Direct Metabolomics from Tissues and Cells

em

Leaf

40x

100x

I'"....q

'" I/) '" I~ ........ or-- MON .... CD~ r; ai ai r--

1co co '" ~ St.... ""<"'l• .,; .l

':l~~t15x

f"i1Root

i i, ; i200 400 600 800 1000

m/z

40

_ 20

J!l§ 0.LJ~J.Lul'-''''---4-iIIIoloMl'''6oUoo,....."""",__~

8 20QQ

~ 10.....

10mm

Figure 6.2. In iva anat i of a French marigold e dling by LEI M . (/efl pallel) In the eprofiling experiment, rna peclra. acquired on 50-~m-diam ter area of the leaf. ~lem androot, rc cal thal (right panel) the organ e 'hibit dramaticall different primary and econdar.m tabolile compo ition. ( dapted with permi ion from erne. P., and erte. . [20071.La cr ablation electro pray ionizalion for atmospheric pre ure, in vi o. and imaging rna s,pcctrometr . Anal. Chem. 79,809) 106. opyright2007 American Chemical ociet .)

metabolic profiling of the root, tem, and leaf regions of a live French marigold(Tagetes pant/a) 'eedling (Neme. and Vertes, 2007). The ob erveu ions have beenassigned to primary and 'ceondary m tabolite . With superficial damage to the liveti u. local analy i nablc inten ity profile to be recorded acro the variousregion of the seedling. For exampl ,the r at primarily generate i n with m/z Iethan 250, th tern yi Id odiated kacmpferoI3-0-(2" 3"-di-p-coumaroyl)-gluc ideor. omc of it tructural i. omer' with an xcepti nail high abundance, wherca theleaf produce variou ion a. igned to photo ynthetic product.

Quantitative LAE 1analy i facilitate' m tabolomic inve tigation . L 1 ion~ignal inten itie from olution of drug tandard indicate that the follow a lin arcorrelation with analyte concentration er a four-decade dynamic r nge ( eme'et al.. 2007). Metabolites in biological 'ample are present in a complex matrix ofconstituent, each of whieh can compete for the available charge within the clec­trosprayed droplets. In omc case, lhi re ull in narrower dynamic range or lim­it· quantitation to thc determination of r lative conc ntration within th ample.Using d uterated analogue a internal tandard, the ab olute concentration' ofsmall mctabolite , including ncurotran mitter and 0 molyte (e.g., ch line, carni­tine. and hetaine), have been identified in the lectrical organ tis ue I' Torpedo cal­ifornica ( ripadi et aI., 2009). ath r report hav e tabti hed quamitation I' lipidsin the mouse brain, allowing the mea urcmcnt of the ab olute concentration of P(34:1) with good analytical performane (, hre lha et al., 2010a). The anal te Ie cIdet rmined by LAE I M have agr u with I vel reponed by traditional anal t­ical method. The ability to correlat ion ignal inten itie with native metabolite

1. Direct TIssue Analysis and Imaging 145

C81 cells

Folds increase

~~vt...L-Ornlthlna PUlrasclene

CO, mil 89

Spo........._+Spermine --- Spermichne

mll203 .,....- mil 148

GuanldlnoaCOlalO

Creatine Phosphocrea1lnemlz 132 c,..... "'"

.,...,...Glyeln

Arglnlna(urea cycle)

mil 175

8642o24

CEM cells

6810

Figure 6.3. (left panel) Metabolic difference between control ( EM) and virally tmn formedT I mph eytes ( 1) identified by LEI MS. The 010 t affect d metabolite were (/) thioac­

tamide. (2) putre cine or pyrrolidine. (3) choline. (4) proline. (5) taurine. (6) creatine. (7)permidine. (8) p-aminob nzoic acid. (9) iminoa partie acid. (10) arginine. (//) dopamine.12) ph phocholinc. (/3) carbamoyl-pho phate. (J.I) pcrminc. (/5) methox. tyramine. (/6)-acetyl a panic acid or N-formyl glutamic acid, (17) homovanillic acid. (/8) glycerophos­

phoch line. (19) glutathione, (20) -hydro yguan sine. and (2l) adeno ine monopho phate.The e change~ pointed to ignificant perturbations in the (top right panel) creatine andp Iyamine bi . ynthe'i' pathway and the (bottom right panel) lipid metab Ii m pathway.

dapted from ripadi, P.. hrc tha. B.. a Ie . R. L.. Carpio. .• Kehn-Hall. K.. he a­i 1', •• et al. [2 10]. Direct detection or diver e metabulic change in virally tran formed and

Ta:-e pres.ingceJl byma pectr metry.PLo ONES,eI2S90.)

nc ntration in ti ue and c lls i ntial in th inve tigali n of metabolic- aog s in an organism.

Human mctab lic pathwa and their chang can also be explored by L If. irally tran formed and Tax-expre ing human cell were proJiled more

r ntl in high-throughput xperiment ( ripadi et aI., 2010). Figur 6.3 pre nt. 0 of veral bi ynthetic pathways ignificantly altered by the viral tran-forma­

n of h t cells. uch mea urement can be perf rmed in a few minute anJ reg uirenl mall cell population. With LAE J M . everal virus typ - pecific (HTLV1 v .

3). expre i l1-specific (TaXI v . Tax ), and cell type- pecific T lymphocyte. kidney cpithelial cells) change were ob'crvcd in the metabolit profile. The eature indicate thaI LAESI M can be u· d in biomark r di covery and pati nt

nitoring. More xtensive invc tigations addrc sing how di ea e states alter cellu­r metabolic pathway can al 0 enable new treatment trategie.

1.3. Metabolic Imaging

I MS offer a mean to achi v I cal analy'i with patial re olution to 50 ~m.

del' appropriat experimental condition (e.g., temperature and humiJity). the

146 Direct Metabolomics from Tissues and Cells

tructural (lnd chemical integrity of bioi gical tissue' and cells i e. sentially retainedaround the ablated p l. Auming proportionality betw en the LAE ] M ignaland the metabolite concentration in the tissue, patial variation in the chemicalcompo ition of the e y tern' can be interr gated. The i n ignal mea. ured aero sthe tissue' allow the recon truction f the corre ponding molecular image. To per­form LA ~ I rna" pectrometry imaging (M T). the ample i mounted onto a am­ple holder (Figure 6.1). ing two computer-controlled independent tran lationtages (X and Y direction ), the sample i po ition d at the focal pint o[ the ablat­

ing mid-infrared light. The generated ion are imultaneou I rna -anal zed, andthe data are stored for each X- Y coordinate o[ th interrogated ar a. Detailed pro­t col of lateral imaging by LAE I M and data evaluation can be found eL ewhere( me and Vertes, 2010a. 2010b). The mol cular image f the ampl [r a partic­ular specie i reconstructed by repr enting the intensity of the related ion 'ignalon a fal'e color cale and correlating it with the c ordinate for very pixel of theinterrogated area.

Mo t plant ti ue are tructurally different fr m li ue found in animals andhuman. [n plant, the waxy cuticle and the rigid c II wall act a natural barrier.for unwanted water 10 in an M ] e periment. In contra t, when animal ti' 'uear di' cted, evaporati e wat rio. i. relativ ly rapid. Becau e wat r rves as theenergy coupling medium in LAES[ experiments, animal ti ue sections ar generallykept cold r fr zen during analy e ( m. et al., 2010: hre tha et al.. 2010a). APeltier-co ling tage equipped with a heat ink and a fan, a hown in Figure 6.1, canmaintain the appropriate conditions [or ev ral h ur ( me and V rte , 20lOb)and en ure that the water content of ti. sue d e not appreciabl change during theLAESI M I time [ram .

Dire t molecular imaging f thin animal ti ue' ction allow the appreciationof their m tabolic organization at atmo pheric condition. In a more recent exam­ple. two-dimensional distributi 0 of mor than 200 di tinctive ionic pecie wereimultaneously mea ured fr rna 100-f,lm-thick coronal rat (Rat/us norvegicus) brain

'ection ( erne et al.. 2010). Among the monitored metabolite' were neurotrans­mitter molecul , uch a "V-amin butyric acid (0 BA) and choline; polyamine','uch a permidine and spermine; and metabolite essential [or chemical energytran fer. including ad no ine and adeno inc monopho phate. Man lipid peciewere also detected, and several of them were identified as P and glycerophos-phoelhanolamine (P ).

U ing high rna . -resolution tim -of-night M in conjunction with LAE 1. itb com s feasibl to deconv lutc th patial distribution of O1etabolit that p -

, an identical nominal rna . Fi ure 6.4 pre ent the i n imag of SA andcholine. which differ only by .8 mDa in their monoi topic mas. and have be nfound to p pulate the ti sue section in a a t1y diff rent mann r. imilar distrihu­tion can b obtained for any detected m/z.

The ma iv data et g nerated in LA I M I can be evaluated via Pearsonco-localization map (em tal., 2010). Figure 6.4 how the ti ue di 'tribution ofth una.. igned lipid ion rnJz 702.537 and the P (37:6) and PE (40:6) on a false-color. calc. The calculated Pear nco-I alizati n map highlight the area' where the. etwo ion are found tog ther. o-localization of metabolite can b exploit d in thedi c very of m tabolic patbwa active in certain ti ue regions.

1. Direct Tissue Analysis and Imaging 147

GABA·__ .·1:'?"W

/"Ja.:' "'\. .... ."'. .... -_ .. -

'104.070

104,05 100.10

mil104.15 100.20

Figure 6.4. Metabolic imaging of a IOO-l1m-thick coronal ection of a rat brain by LA (M.High ma '-re olution differentiates among ion with identical nominal mel es, including theG BA and ch line ion, which e hibit dramatically different patia! di tribution. harac­teri tic ti sue localization is found for evcra) metab lite and lipid, including adcno inc, anunas ign d lipid, and a combination of P (37:6) an<.l P (40:6). The Pear on co-localizationmap b tween the una igned lipid and combination of PC (37:6) and PE (40:6) di,tributi nscan help to explore metabolic relation in pace. cale bar d note 1 mm. ( dapted withp rmi ion from emcs, P.. Woo<.l , A, S., and VerLe , A. [2010j. imultaneou imaging ofmall metabolite and lipid in raL brain ti ue aL atmo pheric pre sure by laser ablati nI clrO pra i nizali n rna pectrometry. Analytical Chemistry 2,9 2-9 . op right 2010merican hemical ociety.)

ari u metabolite can al 0 be imaged in plant tis. ue . Jon ignal regi. t redfrom plant by LIM often c rre pond to primary and econdary metah lite,nd many of them exhibit ti 'ue- pecific accumulation pattern.. Leav of the arie­

:o-ated zebra plant (Aphelandra squarrosa) can erve as an example. orne mctabo­lite uch a kaempfer I-(diacetylcoumaryl-rhamno ide), arc found to be homo­:0- ne u I di tributed in the ti ue, whereas thers, including meth xykaempferol_ ucoronide and chi r phyll a, excJu ively populate the ycllow and grecn s ctor ,

. p ctively ( erne et aI., 200 ). In-depth inve tigati n reveal that th di tribu­n [certain econdary m tabolite arc linked Lo plant phy i logy. For example,Arabidopsis thaliana leave, LAE 1 M I tudie revealed that the outer

rnina and the midvein ynth ize 4-methyl 'ulfinylbutylgluco inolate, indol-3­tb 19luco inolate, and -methyl ulfinyloctylgluc ,inolate in hi h c nc ntrati n

_ me et aI., 2009b). In agreement with the c LA 1 M I result, independ nt

di found imilar concentrati n profLie and ugge t d that they wer con­t nt with a natural plant defense mechanism again. t herbivore attack ( hroff

~ aI..2008).

1. • Metabolic Depth Profiling and Three-Dimensional Imaging

ultiple la er pul'e are delivered to the ame pot on the ti. ue, each pul-eve a con ecuti e layer f cell, hi enable the chemical compo ition of thrlying voxel (volumetric pixel) to be prob d producing a depth profile. The

unt of material sampled by each la 'er pulse i governed by the applied lao I'

o and the l n 'ile trength of the tis ue ( eme et aI., 2008; Nemes and Verte •; erte' et aI., 200 ). Mid-infrared la er ablation bas been d m nstrated withremoval rate between 0 ~m/pul e and 80 ~m/pul e ( em . et al.. 2008:

148 Direct Metabolomics from Tissues and Cells

'000

00'.........,.

• •xImm) " 12 12

'"'14'20.."

Figur 6.5. Thn:e-dimcn ional metabolic imaging in I af ti ue b L E. I . The epider­mal and pali 'ade me oph II region of the palhiphyllwll Iyni e leaf ti' u accumulate (leftpanel) c anidin/kaempferol rhamno ide gluco ide (m/z 595.2) and (middle pallel) chi I' phylla (m/z 893.5) at high level. (righl panel) [n the variegated leave of A. squarro a, acacctin(mlz 285.0) L abundant in the 'econd and third layer of the Ii sue with a homogeneous di ­Iribulion in the other '. ( dapted with pcrmi ion from eme, P., Barton, . ., and crte,A. [2009]. Three-dimen i nal imaging of metabolite in ti ue under ambient condition bla 'er ablation electro pray ionization ma spectrometry. AnalYlical hemiSlry 81,666 -Q675.Copyright 2009 American hemical ociety.)

erne ct aI., 2009a, 2009b). The rc ult of ingle-cell analy i ( hI' tha and V ne.,2009; hre tha et al.. 2011) ugge t that voxel dim n ion can b tailored to I' achsubc HulaI' level .

Three-dimensional M I i I' alized by the combination f lateral imaging withd pth pI' filing ( erne et aI., 2009a), In practice, the ti sue i po itioned lat rallywhile c n ecutiv la I' pul e expl I' the depth ariation in it ch mi try at each(X, Y) coordinate of the interrogated area. Detailed information on in trum ntationand data analy i is available el ewhere ( erne and Vertes, 2010b). In the acquireddata et ,the inten it di tribution f reach m/z i mapped to th ab olute po ition ofthe ox I, ielding th three-dimcn ional molecular image f the ti ue for everydetected m/z ignal.

Re. Ull of three-dimen ional LAE I MSI indicate that primary and secondarymetabolite' often populate plant ti 'ue in a di ·tinctivc manner. For example, inthe peace lily (Spathiphyllum Iynise) leaflet, cyanidin/kaempfer I rhamno ide glu­co ide a fund in the top 40 fJ.m erne et al. 2009a). The corre ponding lhrc ­dimen'j nal di tribution, een in the left panel of Figure 6. -, re eal the leva tedabundance [tm' specie' in the epidermal region of the ti u. In agreement withthese result, indep ndent tudie rep rted higher concentration of kaempferoJ gl ­co ide' in the upp r epidermalla er , likely t erve in part a ultraviolet- creeningpigment· again t the detrimental effect of olar radiation (Alcniu. et al.. 199 ).In contra 't, the middle panel of Figure .5 how that chlorophyll a i abundant inthe palisade mesophyll layer of the leaf tis ue [rom 40 to 80 fJ.m, in agreement withthe known localization of chIor pla·t within plant ti. ues. A different di tributionpattern wa' revealed in the three-dimen ional M I of A. squarro a leave' ( emet al.. 2009a). The right panel of Figure 6.5 pre ents the patial di tribution f

2. Metabolic Analysis of Single Cells

acacetin and indicate - lhal lhi econdar metabolit folio th yell w ctor ofth variegated tis 'ue in the buried 'econd and third layer but doe not exhibit het­r geneity in the top and bottom epidermal layer .

The in-depth inIormation gained by three-dimensional LAESI M I enablem tabolomic tudies on a new level. For example, two-dimen. ional M I h w thatth di tributi n f kaempferol-(cliacetylcoumaryl-rhamno ide) i homogeneou inthe A. quarrosa leaf ti sue (Nemes et aI., 200 ), However, the lhree-dimen jonalon images indicate that thi econdary metabolit accumulate only in the me ­phyll layers ( erne et a!.. 2009a). a piece of information that i 10 t in alllalcral

imaging exp riment that fail to report on the ab olute depth of analysi , The abilityo interrogate biological sy tern. in thr e dim nsion ha far-r aching implications

tn m tabolomics and i likely to boo t I' arch in the life cience .

2~ Metabolic Analysis of Single Cells

, J -cell gen expr s'ion profiling using P R ha ,hown cell-to-cell variability~ ~n within the 'arne cell type ( t' hlberg and Bengt on 2010). las ical ana­_ lca] t 01 , • uch a laser capture microdi ection coupled with microarray . have

V'll h terogeneity f r the gene e pres -ion am ng . ingle neuron in a rat hip­mpu (Kamme et a!.. 200 ). How ver, tandard metabolomic method. u ually

Iyze num rous cells and provide informati n on the average metab lome, disre-ingcell-t -cell heterogeneit . Direct ingl -cell metabolomic require the anal­f chemically div I' e metabolit in a mall ample volume defined by the cell

Ideally, analytical method for direct single-cell metabolomic analy is reyuire'mal ample preparati n and exhibit high en ilivity and acute e1ecti ity. Based

ctro c py, chromato raphy. and M techniques, a diverse array of analyticalruque have been u d for tudying a varying number of m tab lite in sin­

. Excellent review on thi topic are available ( mantonico et aJ.. 2010a;id tal., 20JO; Wang and Bod vitz, 2010). In thi section, we briefly pre nt

t c mm n approache u ed for the metabolic anal si f single cell withI mpha'i on M technique.n cla ical ingle-cell detection and orting lechnique, flow cytometry, can

10 ed for the id ntification and cparation of cells that exhibit a particulart). uch a a di 'ea e tate. In me ca C , floweytom try with antib dy-ba ed

or enetically encoded flu resc nce can be u ed to characteriz a mall num­i hemical in inglc cell (Borland el aI., 200; oh net al.. 200 ).

. trochemical detecti n of m tab lite in ingl cell require' the u of:=::I::roe.ie'ctrochemical d tector (Ewing et aJ.. 1992). The method ha high n itivity

ult in lab I-free d t cli n but is limited t a few easil xidized biochemi-I elf chemical mea ur ment . a microelectrode can be positioned inside or

'ngle cell to det ct a iJy xidiz d biochemical p cie in th cell (Arbault et:. Ewing el al.. 1992: chroeder et aI., 1992: chulte and Schuhmann. 2007).

• 1R i a well-e tabli hed analytical technique for metabolic pr filing of ti ­II extracts (B ckoncrt tal., 2007) ( ee al 0 hap tel' 1 ). More than two

a 0, MR pectro copy all wed the analy i of metabolites in human ery­c 11 population. and MR imaging wa u ed to map the lipid content of anopu laevi egg (Aguayo et al.. 19 6: Brown tal., 1977). 0 molyt and

149

150 Direct Metabolomics from Tissues and Cells

metabolites were tudied more recently in ingle neuron i'olated from the ea 'lug

Aply ia cali/ornica employing MR (Grant tal., 2000; choeniger et aI., 1994).ThL sp ctro copic techniqu can be u d for the lab I-free, nonde tructive, and in

ivo int rr gation of metab lite in the e large ceiL. but it ha in ufficient en itiv­ity to w rk with 'ingl animal cell of more common ize (Griffin, 2006; Kim et al..20LO). Developments in micr trip technology have enabled the NMR detection ofLOO pmol of analyte and can pr vide nough en. itivity to tudy metabolit in mallc II population (Krojanski et aI., 200 ).

Fluore cence-ba ed analytical method in combinati n with micro copy ha

been particularly 'ucc . sful in ·tudying the chemi try of ingJe cell and ubc lIular·tructurc (Muzzey and van Oudenaard n. 2009; Wat on. 19 7: Wu et aI., 200 : Yipand Kurtz, 2002). Th . e technique are nonde tructive. have impre. i e detecti n

limit (down to the sin'le molecule level), are capable of high-thr ughput analy-, and are quantitati e in nature. Flu re ccnce-ba ed metabolomics r quire' the

introduction or presence of fiuorophore inside a cell. For example, u ing coupled!luore cent proteins. the malto level in the cyto' I f liv yea t cell and th glu­co concentrati n. in immortalized kidney c U' were xamln d in ingle-cell fiu ­r cence re onance ncrgy tran. fer (FRET) xperiment (Fehr et aI., 2002, 2003).

ome metabolit ar natively f1uore 'cent and can be directly monitor d withoutchemical tagging. For e ampl , the amount of carotenoids in yea t cells was calcu­lat d ba d n autofluore cence (An et al.. 2000). It rnatively. gen tic encodingfor the pr duction of the f1uorophor (.g., a green fluorescent prot in [GFP]) ispo ·ible.

apillaryelectrophor i ( E) i another power[ultechnique and ha b en cou­

pled to various detector to characterize biochemical 'pecics in single cell (Arcibalet al. 2007). The direct analy i' of . ingle cell by E is typically p rformed bin erting a microcapillary int the c II for ampling. Th extracted material can beanalyzed u ing electroch mical dctccti n, la. er-induced fluorescence, or M ( ruzet aI., 1997; Hofstadlcr et aI., 1995; Hogan and Yeung, 1992; Kenn dy et al., 19 9:Lillard tal.. 19 6; Olefirowicz and wing, 1990; im et al.. 199 ; Yeung, 1999).

Th utilit of combined with E J M for analyzing metabolite in ingle cellha, been demon trated on neuron of A. cali/ornico (Lapainis et aI., 2009; emes

ct aI.. 201 1).The analy i of ingle cells using MS re ults in the imultan OllS det ction of

multiple pecie. M t chnique operate without chemical labeling, can elucidatetruclural information, and, in many ca ,have adequate en 'itivity for single­

cell experiment. With MS, multipl metab lite can be detected with ut elect­ing them before the analysis (Monroe et al.. 2007). How ver. Mia destructi etl;chnique and u ually requires careful sample preparation. Metab lie proliUng habeen achie ed f r extremely large cell ,th X. laelli oocyt . by G -M u ing in­liner ilylation (K ek et aI., 2009). B cau e f th high patial resolution of the ionputtering proce , ec ndary ion ma' pectrometr ( 1M ) ha al 0 been u ed to

analyze m tabolite within a ,ingle c II and in ubc Ilular ·tructure (Chandra ct aI.,

2000: Colliver et al.. 1997; 0 trow ki et aI., 2004).latrix-a si t d la er de orption ionization (MALDI) M ha be n u d to

analyze neuropeptide in inglc neurons (Rubakhin et aI., 2003) and peptides inmammalian cell uch a: ingle rat in term diate pituilar cell (Rubakhin et a1.,

2. Metabolic Analysis of Single Cells 151

Figure 6.6. Compari on of ma p c­tra obtained by the analysi of), 100.400, and 500 cancer edl by laser 1M(btue). ion NIM (green), MALDI(red), and nanoe) ctro pray ionization(black) demon trate the uperior n-iti ity and metabolite coverage of In er'IM . The inset how a fluore cent

image of the cell after laser N 1M anal­. i. The 'cale bar i 2 ~m. (Adapted"ith permi ion [I' m orthen. T. R..

ane . 0., orthen, M. Too Marrin-uc i. D., ritboonthai, Woo Apoll. J .• etal. [20071. Clathrate nano tructurc rarrna pectrometry. Nature 449, 1033-'3. opyright 2007 ature Publi hing

Group.)

mlz

Laser-NIMS

MALOI

900

6: Rubakhin and w edler. 2007). ano tructure-initiator ma sp ctr metry) with la er excitation ha 'hown ensitivity at the ingle-cell level for analyz­

~ eukaryotic microorganisms, yeast (SacchCll'omyce cerevisiae). and single human1 (Amantonico ct aI., 2009: Andrea et aI., 200: orthen et at., 2007). Figure 6.6

w rna spectra obtained [r m 1. 100, 400. and 500 cancer cell (MD -ME­_ 1) b la er- 1M ,ion- 1M . MALDI, and nanoelectr pray ( orthen et aI.,

. The e rna pectra eemed t indicate higher effici ncy to ionize nd ge­phD pholipid and m tabolil in la er 1M xperimcnl than by the compet­

-: m thod . A imilar MALD£-ba. ed technique ha been empl yed to character­m tabolic heterogeneity within a c II population of unicellular ukar tic algacerium acero 'wn (Amantonic ct aI., 201Ob). Direct la er de orption ionizalionha al 0 been used to 'ludy lrongly absorbing s coodary m tabolite' in leave,

nta, tamens. and tyli in plants with single-c II resolution (Dirk el at.. 2009).r recently introduced matrix·free 1a er d orption ionization melhod ba ed

Wcon nanop t arrays ( AP ) ha hown lh abilit to anal ze lh metab ­In ingl yea t cell and v ry mall cell population wilh a high coverage of the

c;;:,~.llllulome(Walker el al., 2011) in vacuum.On direct method to analyz metabolite' in ingle cell uses videomicro. copymbination with M (Mizuno et al., 200 ). a 'ch malic i h \i n in Figure .7. In

proach, content of singl c II arc drawn into a nano pra lip under vid omi­Ji:l"",,(lpy. followed by the addition of an eleclro pray solution to tb ampl d cell

ri I. At thi point, the nano pray tip is converted inlO an electro pray emitter,prayed cell content i analyzed by a rna pcctromcter. This approach ha

d the delection of erotonin and hi tamine in granule of rna t cell, knownical mediator during allergylimulati n.

152 Direct Metabolomics from Tissues and Cells

(c)(a)nanospray tip

cells

• NH,

..-Figure 6.7. Analy i of a ingle cell by video M . (a) The cell are observed by videomi­cro copy. (b) ontents of a cell are extracted into a nano pray ionization tip. The I olventi' cumbined with the cell content in the tip and (c) the mi ture i electro pra ed into themas . pectrometer to obtain a (d) ma pectrum. (Adapted with pcrmi ion from izuno.H., T uyama. ., Harada. T., and Masujima. T. [200 ]. Li single-cell ideo-mao pectrom­etry for cellular and ubc Hular molecular detection and cell cia ification. Journal of MasSpeCTrometry 43, 1692-1700. pyright 200 John Wil yand n, Ltd.)

ingle-cell metabolomic i a particularly pI' mi ing application of LAE J MS.everal operating pararn t f' all w fine-tuning of th amount [material ampled

during mid-infrared ablation [ I' uch purpo c . The pul'e energ and f cu ing prop-rtie f the La I' beam, the ptical and mechanical prop rtie of the ampl ,and th

geom try of th LAE I etup are ju t a few underlying variabl that can help tailorth analyz d area and volume to the pby ical dimen ions of individual c II . ThereLated in trumentation extend' beyond the cope of thi chapter, and the readeri. referred elsewhere ( erne and Verte , 201Oa, 2010b; hre tha and Vert ,2009,2010). Among the available technique for ingle-cell metabolornic b LEI ithe u e fa. harpened optical JIb I' to deliver the lao er pul e to ablate the electedcells. The election of a ingl cell and the c upling f the la r pul e to the c Llare a i ted by two long-di ·tance micro cope. igure 6. how uch an arrange­ment that i u cd to analyze individual epidermal cell of Allium cepa ( hre tha andVerte , 2009). Each cell yielded signal' for more than eventy chemically different

References

100 203.0520

219.0258~ 80 3651067'iiic:S 60.EGl> 40":ll'llQjCt: 20

0150 300 450 600 750

m/z

Figure 6.. Single-cell mctabolic anal ~i of A. cepa epidermal ti- u' by LEI M . (leftpalleT) A chemically etched optical fiber i used t 'clect and ablate the cell of intere l. (rightpanel) MS analy i of the gencrated ion reveals phenotype- pecific cc ndary metabolite

mp ition, includjog cyanidin gluco idc that i abundant in the purple cell of the ti ueInset). (Adapted with p rmi ion fr m hre tha, B.. and Verte, . [20091. In itu metabolicrofiling of ingle cell by la er ablation I ctr pray ionization ma pectromctry. Anal.

Chem.81, 265 271. opyright 2009 American hemical ocicty.)

n .Bytarg tingnumerou cell th metaholitecont nt fneighboringcell'canbeanalyzed. F I' example, it wa found that purple cells contained ignificant I vel. of

nth cyanidin. other navonoid , and thcir gluco ide. including cyanidin gluco ideh.re tha el aI., 2011; hrc tha and erte, 2009). In agr m nt with the e re ult ,

latter i a kn wn purple pigm nt and i. contained in Ihe cell vacuole in purple

I at .xt nding ingle-c II analy i to mall cell populati ns [fer new in ight

z-arding cellular heterogeneity. In a more recent development. thirteen neighbor­_ c U in the epidermal tis ue of A. cepa were ampled for their metabolic compo­i n (Shrestha 1 a1., 2010b). The variance in the data can hed light on c llularI rogeneity n a m tabolie level und I' native-like experimental condition .

. di cu. sed previou Iy. the dir ct m >tab lomic anal i of ingle c II u ingi a quickly emerging field. We anticipate rapid growth in the development of

~ tool and method' for the ambient M analy i. of. ingJ cell and small cellulation..

EFERENCES

_ \0. J. Boo Blackband, . J.. choenigcr, J.. attingly, M. . , nd Hintermann, M. (19 6).u lear magnetic re onancc imaging of a ingl cell. Nature 322, 190-191.mu • C. M., Vogelmann. T. C. and Bornman, J. F. (1995). thrce-dim n ional repre­nlation of the relation hip betv cen penetrati n of u.v.-B radiation and u..- 'crecning;ment in Ica e of l ra iea napu. ew Phytolo iSI 131. 297-302.ntonico, A., Flamigni, L., Glau, R. and Zenobi, R. (2009). egati e modeo truclurc-initiator mas pectrometry for dctection of phD phorylaled metabolite.

(abololllie. 5,346-353.tonica, rban. P. and Zenobi, R. (2010a). nal tical technique for ingle-cell

labolomic: tatc of the art and trends. Analytical Gild Bioanalytieal hemi try, "'98,_ 3-2504.

153

154 Direct Metab%mics from Tissues and Ge//s

mantonico A. rban, P. L., Fagerer, . R., Balabin, R. M. and Zenobi, R. (20LOb). ingl·cell M LDI· as an anal tical 1001 for tudying intrap pulation metabolic heterogeneilyof unicellular organi m . Analytical Chel1li try 2,7394-7400.

An, G. H., uh,O, ., Kwon, H. ., Kim, K. and John on, E. A. (2000). Quantification ofcarotenoid' in cell of Phaffta rhodozyma by aul flu re cence. BiotecJmology Letters 22,1031-1034.ndrea, ., Joo Yeon, 0., Jen, ., Matthia, H. and R nato. Z. (200 ). Ma spectrometricmethod for analyzing metabolite in ea t with ingle cell sen itivity. Allgewandte hell1ieIl1temalional Edilion 47. 5382-53 .

Apitz, I. and Vogel, A. (2005). Material ejection in nano econd Er:YAG la r ablationof water, liver, and skin. Applietl Physics A·Material Science and Proces ing 81, 329­338.rbault, ' .. Pantano. P., Jankow. ki, J. A., Vuillaume, M. and Amat re, C. (1995). Monitoringan oxidalive tre mcchani. m al a ingle human fibrobla. t. Analylical Chemi ·try 67. 3382­3390.

Arcibal, I., antillo. . and Ewin o , A. (2007). Recent ad ance in capillary electrophoreticanaJy'i of individual cell. Anal tical anti Bioanaly/ical hemi try 387, 5J-57.

Beckonert, 0., Kcun, H. .. Ebbel , T. M. D., Bundy, J., H Ime , E., Lindon. J. .. e/ 01.(2007). Metabolic profiling. metab 1 mic and metabonomic procedure for MR pec­troscopy of urine, pIa rna, erum and ti ue extract. alLlre Protocols 2.2692-2703.

Bohrer, B. C. Mererbloom. . I., K enigl:r. . L., Hilderbrand. . E. and Clemmer. D. .(200 ). Biomolecule analy i bin mobility pectr metry. Annual Review of AnalyticalChemistry I, 293-327.

Borland, L. M., Kottegoda, ., Phillip, K. . and Allbritton. . L. (200 ). Chemical analy iof ingle cells. Annual Review of Analytical hemi try 1 191-227.

Brown, F. F., Campbell, I. D., Kuchel. P. W. and Raben tein D. C. (1977). Human erythro­cytc metaboli m tudie by 1H .pin echo MR. FEB Leuer 82,12-16.handra, .. Smith, D. R. and Morri on, G. H. (2000). ubcellular imaging by dynamic 1Mi n microsc py. Analylical hemislry 72, I04A-I 14A.

hen, H, W., Venter, A. and ooks, R. G. (2006a). xtractive electro pray ionization f rdire t analy'i' of undiluted urine, milk and other complex mixlure wilhout ample prepa­ration. Chemical Comnlllilicalion ,2042-2044.

hen. H. W., Wortmann. ., Zhang. W. H. and Zen bit R. (2007). Rapid in vivo fingerprint­ing r non olatile compound in breath b e tracliv electro pray ionization quadrupolelime-of-flight rna pcctrom try. Allgewnlldte lremie-/l1Iernalional EdiliofJ 46, 5 0-

hen, Z" Bogaerts, A. and Verte • A. (2006b). Pha.~e explo ion in almo pheric pre ureinfrarcd la er ablation from water-rich target.. Applied Phy ic Lefler 9. 04J 503.

hen, Z. Y. and Verte , A. (200). arly plume expan ion in almo pheric pre. ure midin­frared la er ablation of water-rich target. Phy 'ical Relliew E 77,036316.dy, R. B., Laramee, J. A. and Dur't H. D. (2005). er atile new ion ource f r the anal­

i of material in open air under ambient condition. Allalytical Chemi Iry 77. 2297-2 02,hen, D., Dickt:rson J. A.. Whitmore, . D., Turner, E. H., Palcic, M. M., Hind gaul, 0 ..

el al. (200 ). Chemical cytometry: nuorc ccnce-ba, cd 'ingle-cell analy i . Annllal Revielvoj"Analytical Chemistry I, 165-190.

ollivcr, T. L., Brummel. C. L.. Pachal ki, M. L., wanek. F. D., Ewing, A. G. and Win ­grad. . (1997). Atomic and molecular imaging at the inglc-c lJ level with TOF- 1MAllalyli 01 hemisrry 69, 2225-2231.

ruz, L., Moroz, L. L., Gillette, R. and weedier, J. . (1997). itrite and nitrate level inindividualmollu can neuron: ingle-cell capillary electrophore i analy i . Journal of C/l­

rochemistry 69, 110-115.Ewing. ,G., trein. T. G. and Lau. Y. Y. (1992). Anal tical chemi try in microenvironment:

ingle nerve cell. Accollnt of hel7lical Re earch 25..w~7.

References 155

F hr, M., Fr mmer, W. B. and Lal nde, . (2002). Visualization of malta uptake in living)ea t cells by nu re cent nanosen.or . Proceeding.s oJthe National Academy oj Science ojthe United tate' oj America 99. 9 46-9 51.

F hI', ., Lalonde, " Lager, I., W Iff, M. W. and Frommer, W. B. (2003). In vivo imaging ofth d namic' of glucose uptake in the cyto 01 of 0 -7 cell b nuore cent nano.en ors.Journal oj Biological hemi try 27 , 19127-19133.

Grant, . C., Aiken, . R., Plant, H. D., Gibbs, ., Mareci, T. H., Webb, A. G.. et 01. (2000).'\ilR pectro c p of ingJ neuron.. Magnetic Re 'onallce in Medicine 44. 19-22.

Griffin. J. L. (2006). The Cinderella tory of metabolic profiling: doe metabolomic get to goto the functional g nomic ball? Philo ophical Transaction ojthe Royal Society B: Biolog­tal dence 361, 147-161.

H I cher D., hI' ff R., Knop K., Gott chaldt M., receliu A., chneider B., Heckel,D. G., chubedrt, U. S. and vatos. A. et 01. (2009). Matri -free UV-Ia er de orp­ti nJionization (LDI) rna spectrometric imaging at the ingle-cell Ie el: di. tributi n of

ondary metab lite of Arabidop i thaliana and Hypericum pecic. The Plant Journal.907-918.

H f tadler, . A., wanek F. D., Gale, D. C, wing . G. and mith. R. D. (1995). ap-lllary electrophorcsi -electro pray ionization Fourier transform i n cyclotron rc onancem pectrometry for direct analy 'is of cellular protein. Analytical Chell7i try 67, 1477­1 O.

H n, B. L. and Yeung, E. . (1992). Determination of intracellular pecie at the levelf 3 ingle erythroc t via capillar electroph re 'i' with direct and indirect nu rescence

d t clion. Analytical hemistry 64. 2841-2845.mmc, F., Salunga, R., Yu, J., Tran. D.-T., Zhu. J., Luo. L.. et al. (200~). ingle-cell microar­

r ) analy i in hipp campu C I: demon tration and validation of cellular heterogeneity.J L1malof ellroscience 23. 3607-3615.nn d , R. T.. Oate , M. D., opel', B. Roo ickerson, B. and Jorgen on, J. . (19 9).· licrocolumn eparation and the analysis of ingle cell. Science 246, 57-63.

,. m. H. K., hoi, Y. H. and Verpoonc, R. (2010). NMR-based metabolomi analy'is ofplan . Nature Protocols 5, 536-549.

, M. Moo Bakel F.. Engel. Woo an den Maagdenherg, _. Ferrari. M. Doo ulier, L.,tal. (2009). Metab Lic profiling of ultra mall. ample olum . \ ith G 1M : from microliter

· nan liter ample. Analytical hemi try 2.156-162.mberg, H. (2000). Kreb and hi trinity of c cJc . Nature Reviews Molecular ,/I Biology· 225-228.

Jan ki, H. G., Lambert, J. R., Gerikalan, Y.. uteI'. D. and Hergenroider, R. (2008).Ii 1'0 lot MR probe for metabolomic tudie. Analytical hemi try 0, 6 672.

In' , T.. Rubakhin . . and \ eedler, 1. V. (2009). Capillary electroph re i. with elec-tr pray ionization mas. spectrometric detection for ingle-cell metabolomic . Analytical

hemistry 81,5858-5 64.'I., hrestha, B. and Vertes, A. (2007). Atmo phcric pres ure molecular imaging byrared MALDI ma s spectrometry. Analytical hemistry 79. 523-532.rd, . J.. Yeung, .. and IcCio ke ,M. . (1996). Monit ring exoc. to i and relea e

r m individual rna t cell b capillary electrophore i with La er-induced native fluore ­nc detection. nalyticnl hemistry 6 ,2 97-2904.

!Zuno H., T uyama. ., Harada, T. and Masujima, T. (2008). Live ingle-cell vide -masctrometry 1'01' cellular and 'ubcellular molecular detection and cell c1as ification. Journal

f'vlas Spectrometry 43, 1692-1700.nr e, E. B., Jurchen. J. Coo Rubakhin, . . and weedier. J. . (2007). ingle-cell mea­ur ment with rna pectrometry. ew Frontiers in Ultrllscllsitive Bioaflalysi : Advanced

.lflalytical Chemistry Applicat;on in Nanohiotcc!mology, Single Molecule Detectioll, andIII Ie Cell Allalysis (ed X.-H. N. u). pp. 269-293. J hn Wiley and on. Hob ken.

uzzey. D. and van Oudenaarden, A. (20 9). Quantitative time-lap e nu re cencei 1'0 cop in ingle cell _A 111//101 Review oj ell and Developmel/tal Biology _5. 01 -327.

156 Direct Metabolomics from Tissues and Cells

erne. P.. Bart n. . A .. Li, Y. and erte, . (200 '). mbiem molecular imaging and depthprofiling of liv ti ue by infrared lacr ablation electro pra ionizalion mas pcctrometr.

Ilulytical hemistry O. 4575~ - 2.eme , P., Barton. A. A. and rte. , . (2009a). Three-dimensional imaging of melab litein Ii . ue under ambient condition b la er ablation electro pra ionization mas pee­trometry. Analytical hemi try 1,666 -6675.

eme., P., Huang. H. H. amI Verte ,A. (2012). Internal energ dep sition and ion fragmen­tation in atm pheric-pre ure mid-infrared la or ablation eleClr pray ionization. Phy. ical

hcmi rry Chemical Phy ic 14.2501-25 7.m s, P. Knolhoff, A. M., Rubakhin. . . and weedier, J. V. (2011). Metab lie differentia­tion of neuronal phenotype by ingle-cell capillary clectrophore is electrospray i Ilizationma pectrometry. Analytical he1l1istry 83, 6810-6 17.eme , P., Margillean, I. and Yerle " . (2007). praying mode effect on droplet formationand ion chemi try in electro pray. Analytical hemistry 79, 10--3116.eme P., vatos, A. and Yerte, . (2009b). Ambient ma pectrometric imaging I'metabolite' in MLI muscllllI brain and Arabidopsi thaliana leaf by mid-infrar d la erablation electro pray ionization. 57th A M Conference on Ma pectrometryand lIiedTopic. Philadelphia, P . p. 141.eme:, P. and erte. A. (2007). La er ablation electro pray ionization for atmospheric pre -

ure, in vi 0, and imaging ma p ctrometr. llat.\IIical Chemi try 79, 09 106.I eme • P. and rte, ,(2010a). lmo ph ric-pr . ur molecular imagin of bi logical

ti ue and biofiJm by LEI rna p etromctr.. Joumaf of Vi ualized Experilllems 4 ,hllp:l/www.jove.com/inde·detail".tp?id=2097.

eme . P. and Verte . A. (2010b). La r ablation electro pra ionization for almo ph ricpr ure molecular imaging rna s pcctr mctry. In: Mas pectrometry lll1aging: Principleand Protocols, pp. 159-171. pringer, Heidelb rg.emc', P. and Verte , A. (2012). Ambient rna~ speetr rnetry for in vivo local analysis and in

itu molecular ti ue imaging. Troc- Trend in I\nul.Vlical hemistry 34, 22-34.erne, P.. Woods, A. S. and Yert-., A. (2010). imullaneou imaging of small melaboliteand lipid. in rat brain tis ue at atmo pheric pre ure by laser ablation electro pray ioniza­tion mas pectrometry. Analytical hemistry 82,9 2-9 .

orthen, T. R., Yane ,0., orthen, M. T., Marrinucci, D., ritboonthai, W.. Apon, J., et af.(2007). lathrat nano tructure f r ma p ctrometry. alllre 449, J033-1036.

Olefirowicz, T. . and Ewing, A. G. (1990). apillary electrophore i in 2 amJ 5 mmdiameter capillarie : application to c topla. rnic anal i. Anal tical Chemistry 62, I 72­l 76.trow ki. . G., an B L1. C. T., Winograd. . and wing, A. G. (2004). Ma . pectrometricimaging of highl curved membrane during tetrah mena mating. cience 305.71-73.

Rubakhin. . ., Churchill, J. D., Greenough. W. T. and weedier. J. V. (2006). Profilingignaling peptide in ingle mammalian cell u ing ma pectrometry. Analytical hell1istry

7 .7267-7272.Rubakhin, . .• Greenough. W. T. and weedier, J. Y. 2003). patial profiling with M LDt

M : di tribution of neuropeptide within 'ingle neur n . Analytical Chemi tr 75.. 3~5380.

Rubakhin, . . and weed! r, J. Y. (2007). haracterizing peptide in individual mammacell u ing ma spectrometry. Na/ure Protocols 2, 1987-1997.

chmid, A., Kortmann. H., Dittrich, P. . and Blank, L. M. (2010). hemical and bioisingl cell analy i . Current Opinion in Biotechnology 21, 12-20.

choeniger, J.., Aiken, ., H u. E. and Blackband, . J. (1994). Relaxation-time afu ion M R miero copy of ingle neuron. Journal of Magnetic Resonance, erie B261-273.

chro d r, T. J., Jankow ki, J. ., Kawagoe. K. T .. Wightman. R. M., Lefrou,matore. . (1992). nal i. of diffu ional br adening of ve icular packet of

eholamine rei a ed from biological cell during e ocytois. Analytical077-30 3.

References 157

. H., Beech, I., er al.pectrometry f I' direct

Spccrromerry 19, 701-

uJt. . and chuhmann, W. (2007). ingle-cell micr electrochemi Ir . AngewandreCltemie Inremational Edirion 46, 8760-8777.

• a, J., Huang, M. Z., H u, H. J., Lee, C. Y., Yuan,2005). lectro pray-assi ted laser de orption/ionuation rna

ambient analy i of olid. Rapid omll1unicatiolls il1 Ma

le . A., me, P., hre tha, B., Barton, ., Chen. Z. Y. and i, Y. (200 ). Molecularunaging by mid-l R laer ablation rna pectr meLry. Applied Physic A-Materials cienceoIlId Proce in 9 . 8 5-891.alker, B. N., Ant nakos, " Rettercr, . T. and Verte , A. (2011). Metabolomic l' mall

11 population and ingle cell. of Saccharomyces cerevisiae on a nan photonic i nizationlalform. Submitted.

ang. D. and Bodo irz, . (2010). ingle cell analy i : the ne\ frontier in omic . Trend inBiotechnology 2 ,2 1-290.a on, J. . (19 7). QuantiLation of molecular and cellular probe in population ofingle

IJ U! ing ftuore Cence. MoleClllar anti Cellular Probes I, 121-1u, J. Q., Mc ormick, . D. and Pollard. T. D. (2008). ounting protein in livin o cell byquantitative flu rescence micro copy wilh internal tandard . Biophysical Tools for lJiolo­

• r , Vo12: In Vivo Technique. EI cvier Academic Press, an Diego.

.. stha, B., COles, P., azarian, J., Halhoul. Y.. Hoffman. . P. and Verte , A. (20IOa).Direct anal i f lipid and mall metab lite in mou e brain ti ue by P IR-M LDIand reactive I rna pectrometry. Analysr 135. T 1-75 .

tha. B. eme, P. and Verte, . (201Ob). blati nand analy i or mall cell popula­and ingle ell by consecutive la er pul es. Applied Physic 1\ -Marerials Science and

Proces ing 101,121-126.tha, B., Patt, J. M. and Vene , A. (2011). In itu cell-hy-cell imaging and analysi of

mall cell populati n by mas pectrometr. Analytical hemi try 3.2947-2955.lha, B. and Verte.. . (2009). In itu metabolic profiling of. ingl' cell by la er ablali ntro pra i ni7ation rna pectrometr. Analyrical ChemiJtry I, 265- 271.

ilia. B. and erte, . (2010). Direct analy i f ingle cell by ma pectrometry atmo phcric pre ure. Journal of Vi Ilali:ed Experiments -D, http:// ww.jove. om/indctail . tp?id=2144.r. R., Vergara, ., Muck, A., vato, A. and G r henzon, J. (200). onuniform di. tribu­n of gluc inolate' in Arabidopsis rhaliana leave ha important con equences 1'01' plantfen e. Proceeding of rhe Narional Academy of Sciences of rile Vnired Stare of America,6196-620 I.

. C. E., Meredith, . D., Kra iva. T. B., Bern. M. W.. Tromb rg. B. J. and Allbritton,. L. (199 ). La er-micropipet c mbinati n for ingle-cell analy i.. Analyrical hemi.\rry

- ,4 '70-..rn, K. F., ggio. R. B. M., an l-Ioutte, J. R. and Villa ·B as. . G. (2010). nal tical

Iatform for metahol me analysi of microbial cell u ing methyl chloroformatc deriva·uzation followed by ga chromatography-rna'. spectrometry. NatLire Prorocol' 5, 1709­1--9.

adi, P.. a7arian.1.. Hathout. Y.. Hoffman, E. P. and VeTte. . (2009). In vitro analy­of metabolite from rhe untreated ti ue of Torpedo califomica electric organ b mid­

CraTed la er ablation electr pray ionization rna 'pectrometry. Metabolomin 5, 2 3­2-6.

adi, P., hre lha, B.. Ea'ley, R. L.. arpio. L., Kehn-Hall, K., hevalier, "er al. (2010).Dir cl detection f diver e metaholic hange in virally tran formed and Tax- pre ing

Us by mas p 'ctr metry. PLoS ONE 5. e12590.• hlberg, A. and Bengt son, M. (2010). ingle-cell g ne expre si n profiling using reversetran cription quantitative real-lime P R. Merhod 50,282-2 .'at , Z., Wi man. J. M.. Gologan, B. and ooks, R. G. (2004). Ma . spectrometry am·ling under ambient conditions with de 'orplion electro pra. ionization. Science 306, 471-~

158 Direct Metabolomics from Tissues and Cells

Ye, B. ., Zhang. Y., Yu, H., Yu, W. B., Liu, B. H., Yin. B. ., et al. (2009). Tim -re olvedlran criptome analy is of Sacil/II. IIbtilis rc ponding lo valine. glulamate, and glulamine.PLo ONE 4, e7073.

Yeung, E. S. (L999). ludy of single cell by using capillary electrophore is and nalive fiuo­re cence deteclion. Journal oj hrOI7lQtOgraphy A 30,243-262.

Yip, K. P. and Kurtz, I. (2002). onfocal f1uore cence micro copy mea urement of pH andcalcium in living cell. Cell Biological Applications oj ConJocal Microscopy, Second Edi­tiOIl, pp. 417-427. cademic Pre , San Diego.