Rapid and Selective Screening for SulfhydrylAnalytes in Plasma and Urine UsingSurface-Enhanced Transmission Mode DesorptionElectrospray Ionization Mass Spectrometry

Joseph E. Chipuk,† Michael H. Gelb,‡ and Jennifer S. Brodbelt*,†

Department of Chemistry and Biochemistry, University of Texas at Austin, University Station A5300,Austin, Texas 78712, and Department of Chemistry and Biochemistry, University of Washington,Seattle, Washington 98195

Nylon mesh substrates were derivatized to includeVICATSH, a biotinylated reagent that contains both aphotolabile linking group and a thiol specific captureagent. The enhanced mesh substrates were then usedto capture sulfhydryl analytes directly from urine andplasma samples via covalent reaction between thereactive thiols of the analytes and the iodoacetaminylunit of VICATSH. Photocleavage of the labile linker wasfollowed by direct analysis of the mesh surface viatransmission mode desorption electrospray ionization(TM-DESI). This chemoselective capture method pro-moted enrichment of sulfhydryl analytes and reducedmatrix interferences, thereby resulting in increasedanalytical performance of surface enhanced TM-DESI-MS when compared to standard DESI-MS. The presentwork describes the manufacture of the derivatizedmesh substrates and the quality control assessmentsmade during the manufacturing process, the optimiza-tion of the chemoselective capture method, and resultsof experiments pertinent to biological applications.Integration of the chemoselective capture materialswith ambient ionization and tandem mass spectrometryresults in a powerful combination of speed and selec-tivity for targeted analyte screening.

Chemoselective and affinity-based capture methods are samplepreparation techniques used throughout chemistry, biochemistry,and molecular biology to recover targeted analytes of interest fromcomplex matrices, thereby increasing analytical specificity andsensitivity through analyte enrichment and the reduction ofinterferences.1-4 For example, medical diagnostics and proteomicsstudies routinely employ affinity-based capture methods to inves-tigate the relationship between antibodies and antigens, oftenresulting in the development of detection assays for metabolic

biomarkers, peptides, and proteins.1,2 Similarly, metabolite enrich-ment by tagging and proteolytic release (METPR) utilizes chemose-lective probes to capture and covalently conjugate small moleculemetabolites containing targeted functional groups to solid-phaseresins.3,4 In some capture methods, such as western or Southernblotting techniques, the captured analytes are physically trans-ferred to a secondary nitrocellulose surface for analysis.5,6 In othercases, including METPR and various affinity chromatographymethods, a secondary liquid extraction step is used to reintroducethe captured analytes to solution for standard LC-MS analysis.3,4,7

In either of these scenarios, extraction of captured analytes fromthe affinity or chemoselective substrate to a secondary surfaceor solution inevitably results in some analyte loss; therefore, thereis continued interest in developing methods to analyze capturesurfaces directly (i.e., without additional extraction steps.)

Fluorometry, surface plasmon resonance, and mass spectrom-etry are the detection methods most often utilized for directanalysis of capture surfaces. Due to its impressive sensitivity,enzyme-linked immunosorbent assays (ELISA) and other im-munological assays rely primarily on fluorometry.8 In thesemethods, which are often developed in multicomponent arrays,detection antibodies containing fluorophore tags are used toidentify antigens that have been sequestered by surface-boundcapture antibodies.8 An increase in fluorescent signal is thendirectly correlated to the extent of binding for a specific antigenor set of antigens. Similarly, surface plasmon resonance (SPR)can be used to detect the binding of specific analytes to specializedsubstrates via the alteration of the refractive index of the surface.9

Once the SPR analysis is complete, captured molecules can beeluted from the substrate and subsequently analyzed via electro-spray ionization-mass spectrometry in a technique deemed bio-molecular interaction mass spectrometry (BIA-MS).10 In contrastto BIA-MS, direct mass spectrometric analysis of the capturesurface typically utilizes desorption/ionization on porous silicon

* To whom correspondence should be addressed. E-mail: jbrodbelt@mail.utexas.edu.

† University of Texas at Austin.‡ University of Washington.

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(2) Guillo, C.; Roper, M. G. Analyst 2008, 133, 1481–1485.(3) Carlson, E. E.; Cravatt, B. F. Nat. Methods 2007, 4 (5), 429–435.(4) Carlson, E. E.; Cravatt, B. F. J. Am. Chem. Soc. 2007, 129, 15780–15782.

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10.1021/ac100242b 2010 American Chemical Society4130 Analytical Chemistry, Vol. 82, No. 10, May 15, 2010Published on Web 04/19/2010

(DIOS),11 surface-enhanced affinity capture (SEAC),12 self-as-sembled monolayer desorption/ionization mass spectrometry(SAMDI),13,14 or, more commonly, surface-enhanced laser de-sorption ionization (SELDI).15-28

While it has been explored most prominently in the field ofproteomics, SELDI is theoretically applicable to nearly anyapplication. In the SELDI process, a surface is modified with anaffinity-based probe designed to capture either a specific moleculevia antibody-antigen interactions or a broader class of moleculessuch as bacteria or microorganisms. To complete the analysis,surfaces containing the captured analytes are rinsed to removeinterfering substances, introduced into the vacuum region of amass spectrometer, and subjected to laser desorption ionization(LDI) either directly or following the addition of a suitable matrix(i.e., MALDI). Surfaces for SELDI-MS have taken a variety offorms, including polyvinylidene difluoride (PVDF),20,21 dextran,22

polyethylene,23 and polyester.24 One particularly effective SELDIsurface which utilizes immobilized metal affinity chromatography(IMAC)15,16,25-28 for the selective capture of histidine-containingor phosphorylated peptides and proteins has been commercialized,and the use of IMAC SELDI biochips has been reported innumerous studies, especially those targeting post-translationalprotein modifications25,26 and disease biomarkers.27,28

Recent developments in ambient ionization methods, such asdesorption electrospray ionization (DESI)29-31 and direct analysisin real time (DART)32 have facilitated a new era of high throughputmass spectrometry, where samples can be analyzed in their nativeenvironment and analysis times are typically only seconds persample. While the speed of these techniques is among the primaryadvantages, the elimination of the chromatographic separationafforded by GC-MS and LC-MS typically results in decreased

specificity and ion suppression for low concentration species.While some selectivity can be regained via the careful choice ofthe desorption electrospray solvent30 or via the introduction ofreagents to facilitate ion/molecule reactions with analytes ofinterest,33-35 these methods are not universally applicable, andthus, it is generally recognized that specificity and/or sensitivityare compromised for the dramatic increase in analytical speedthat is gained through ambient ionization mass spectrometry.

The results presented here demonstrate that the increasedselectivity afforded by analyte capture can be merged with thehigh-throughput capabilities of ambient ionization mass spectrom-etry via the utilization of mesh substrates specifically designedto both chemoselectively capture target analytes from solutionand integrate seamlessly to transmission mode desorption elec-trospray ionization mass spectrometry (TM-DESI-MS).36-38 Whilebroadly applicable, the present study focuses specifically onsulfhydryl analytes, which are prevalent in pharmaceuticals andhuman metabolism.39-41 Compared to highly basic or acidicmolecules, such as amines and carboxylic acids, sulfhydryls aremore difficult to ionize via electrospray ionization and are generallysuppressed by easily ionized matrix interferences, thereby makingthem a suitable set of analytes to demonstrate the benefits of theapproach for high-throughput screening applications. This workdescribes the construction and development of sulfhydryl specificmesh substrates using readily available polyamide mesh materialsthat are partially hydrolyzed and further derivatized with neutra-vidin to create a binding surface for VICATSH, a biotinylatedsulfhydryl capture agent containing a photolabile linker.42,43

In addition, results are presented for the capture and analysis ofsulfhydryl analytes directly from complex biological matrices suchas plasma and urine.

EXPERIMENTAL SECTIONChemicals and Materials. Hydrochloric acid, sodium hy-

droxide, methanol, acetonitrile, and water (HPLC grade) werepurchased from Fisher Scientific (Hampton, NH). N-Acetyl-L-cysteine, captopril, cysteamine, 6-mercaptopurine, MES hydrate,penicillamine, 4-[2-[(cyclohexylcarbonimidoyl)amino]ethyl]-4-me-thylmorpholinium p-toluenefulfonate (CMC), and phosphate-buffered saline were purchased from Sigma Aldrich (St. Louis,MO). Neutravidin (a deglycosylated form of avidin) and fluores-cein isothiocyanate (FITC)-derivatized neutravidin were purchasedfrom Pierce Biotechnology (Rockford, IL). Synthrapol dying

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(12) Hutchens, T. W.; Yip, T. T. Rapid Commun. Mass Spectrom. 1993, 7, 576–580.

(13) Mrksich, M. ACS Nano 2008, 2 (1), 7–18.(14) Patrie, S. M.; Mrksich, M. Anal. Chem. 2007, 79, 5878–5887.(15) Nelson, R. W. Mass Spectrom. Rev. 1997, 16, 353–376.(16) Tang, N.; Tornatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2004, 23,

34–44.(17) Vekey, K.; Telekes, A.; Vertes, A. Med. App. Mass Spectrom. 2008, 379–

406.(18) Petricoin, E. F.; Liotta, L. A.; Liebler, D. C. Prot. Cancer Res. 2005, 117–

131.(19) Lomas, L. O.; Weinberger, S. R.; Marks, R. S. Handb. Biosens. Biochips

2007, 2, 885–894.(20) Hillenkamp, F.; Strupat, K.; Karas, M.; Eckerskorn, C.; Lottspeich, F. Anal.

Chem. 1994, 464–470.(21) Vestling, M. M.; Fenselau, C. Anal. Chem. 1994, 66, 471–477.(22) Brockman, A. H.; Orlando, R. Rapid Commun. Mass Spectrom. 1994, 10,

1688–1692.(23) Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843–848.(24) Wang, H.; Tseng, K.; Lebrilla, C. B. Anal. Chem. 1999, 71, 2014–2020.(25) Suen, S.-Y.; Liu, Y.-C.; Chang, C.-S. J. Chromatogr. B 2003, 797, 305–319.(26) Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope,

M. R.; Borchers, C. H. Anal. Chem. 2002, 74, 3429–3433.(27) Tetsuyuki, A.; Takao, Y. Proteome Sci. 2009, 7, 14–17.(28) Hogstrand, C.; Balesaria, S.; Glover, C. N. Comp. Biochem. Physiol. B. 2002,

133, 523–535.(29) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306,

471–473.(30) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311,

1566–1569.(31) Takats, Z.; Wiseman, J. M.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 1261–

1275.(32) Cody, R. B.; Laramee, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 2297–

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(33) Huang, G.; Chen, H.; Zhang, X; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2007,79, 8327–8332.

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(35) Wu, C.; Ifa, D. R.; Manicke, N. E.; Cooks, R. G. Anal. Chem. 2009, 81,7618–7624.

(36) Chipuk, J. E.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2008, 19, 1612–1620.

(37) Chipuk, J. E.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2009, 20, 584–592.

(38) Chipuk, J. E.; Gelb, M. H.; Brodbelt, J. S. Anal. Chem. 2010, 82, 16–18.(39) Srinivas, N. R.; Mamidi, R. N. V. S. Biomed. Chromatogr. 2003, 17, 285–

291.(40) Głowacki, R.; Bald, E. J. Liq. Chromatogr. Relat. Technol. 2009, 32, 2530–

2544.(41) Kuowacki, R.; Bald, E. Anal. Bioanal. Chem. 2006, 385, 855–860.(42) Bottari, P.; Aebersold, R.; Turecek, F.; Gelb, M. H. Bioconjugate Chem.

2004, 15 (2), 380–388.(43) Lu, Y.; Bottari, P.; Turecek, F.; Aebersold, R.; Gelb, M. H. Anal. Chem.

2004, 76, 4104–4111.

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detergent was purchased from Dharma Trading Co. (San Rafael,CA). Polyamide mesh sheets (nylon-6,6) with a strand diameterof 125 µm encompassing an open space of 190 µm were purchasedfrom Small Parts Inc. (Miramar, FL). VICATSH, a biotinylatedmolecule incorporating an iodoacetaminyl group for the selec-tive capture of sulfhydryls and a photolabile o-nitrobenzyllinkage between the biotin and the capture agent was synthe-sized in the laboratory of Dr. Michael Gelb at the Universityof Washington using previously reported procedures.42 Thestructures of the sulfhydryl analytes investigated in this workare shown in Figure 1.

Manufacture of Enhanced Mesh Materials. Scheme 1summarizes the steps involved in manufacturing the surface-enhanced meshes. Mesh materials were marked, cut into 1 cmsquares, and cleaned thoroughly with methanol to remove anyresidual ink. The mesh pieces were then sonicated in an aqueoussolution of Synthrapol (1%) to remove any remaining surfacecontaminants. Following a thorough rinse with deionized H2O,the cleaning procedure was completed by sonicating thematerials in an acetonitrile and water solution (50:50 v:v) for 5min. Batches of cleaned materials were stored in HPLC-gradeH2O for future use.

Acid-catalyzed hydrolysis of polyamide materials to exposecarboxyl groups was performed by submerging the mesh piecesin a 3 N solution of HCl for approximately 24 h at roomtemperature. Following hydrolysis, mesh materials were rinsedwith HPLC grade H2O to remove any residual acid, blown drywith compressed air, and stored dry in a sealed PTFE container.

Derivatization of the hydrolyzed mesh materials with neutra-vidin was performed using a two-step procedure for carbodiimide-mediated coupling of surface carboxyl groups to the primaryamines of the protein. In the first step, the free carboxyl groupsof the hydrolyzed mesh were reacted with a room temperaturesolution of CMC (108 mM) in MES (50 mM) buffer (pH ) 6) for15 min. After rinsing the mesh materials in phosphate-bufferedsaline (pH ) 7.4), neutravidin (either in its FITC derivatized ornative form) was coupled to the mesh materials by immersion ofthe meshes (10 per 5 mL batch) for at least 2 h in a 70 µg/mL(1.2 µM) solution of the protein in PBS (pH ) 7.4). Followingprotein derivatization, the mesh materials were rinsed free ofexcess protein with additional PBS and immersed in 5 mL of a 1µg/mL solution of VICATSH for at least 2 h in the dark. Thebiotin group of VICATSH binds to neutravidin, thus anchoring

VICATSH to the mesh, while the iodoacetaminyl capture agentof VICATSH remains available to react specifically with analytescontaining free thiol groups (Figure 2).

Mass Spectrometry. A one-dimensional automated scanningOmni Spray ion source (Prosolia, Inc., Indianapolis, IN) wasmounted to a Thermo Fisher Scientific LTQ XL (Thermo FisherScientific Inc., Waltham, MA). A prototype TM-DESI adaptor kitalso manufactured by Prosolia Inc. was used to convert thestandard Omni Spray 1-D scanning source to a customized sourcemore suitable for TM-DESI. The adaption included a slottedsample holder in place of the conventional slide holder and theuse of custom sized TM-DESI mesh holders. The mesh was heldin place by affixing it to a 26 mm × 76 mm × 3 mm PEEK backingplate with an 8 mm × 38 mm slot cut into it to facilitatetransmission of the electrospray through both the sample and thebacking plate. In addition, adaptation included a modified sprayarm that facilitated a 0° angle between the electrospray tip andcapillary inlet to the mass spectrometer and an extended heatedcapillary. All analyses were conducted at a distance of 2 mmbetween the electrospray tip and the mesh surface and a distanceof 6 mm between the mesh sample and the capillary inlet of themass spectrometer. Methanol at a flow rate of 10 µL/min wasused as the electrospray solvent and nitrogen at a pressure of110 psi was used as the nebulizing gas. The electrospray voltagewas set to 4.0 kV, the ion accumulation time set to 100 ms andsignal averaging set for three microscans. All mass spectra wereacquired in the positive ion mode by scanning the sample at arate of 250 µm/s. Collisionally induced dissociation spectra wereacquired using an isolation window of 1 Da, a q value of 0.25, andan activation time of 30 ms. Collisional energy ranged from 20 to35 arbitrary units and was optimized for each ion.

Fluorescence Microscopy and Fluorometry. Analysis of thedistribution of fluorescently labeled neutravidin on derivatizedmesh materials was performed using the 2.5× objective of anOlympus BX2 epifluorescent microscope equipped with a 12 bitCCD camera (DVC Co., Austin, TX) and high-pressure mercurybulb excitation source. Excitation of the fluorescein isothiocyanatetag occurred at 480 nm and emission was monitored at 535 nm.Photomicrographs were captured via DVC software with adjust-able gain, offset, and exposure time.

The reproducibility of polyamide hydrolysis and neutravidincoupling was monitored through several fluorometric assays. Allassays were performed on a Perkin-Elmer Victor 3 fluorometerequipped to read samples presented in 24-well plates. Aqueoussamples (1 mL) were deposited in the plate wells, and the analysistime was optimized to provide maximum fluorescence intensityfor positive control samples while maintaining minimal signalintensity from control blanks. Experiments involved fluoresceinor fluorescein isothiocyanate, thus excitation and emission weremodulated using filters of 485 and 535 nm, respectively. To assesspolyamide hydrolysis, representative hydrolyzed mesh materials(five per hydrolysis batch) were weighed and each submergedin 1 mL of an aqueous fluorescein sample (1 µM) and allowed toequilibrate for 10 min. The fluorescence intensity of the solutionwas then measured for 100 ms, and mass corrections were appliedto account for any differences in mesh surface area. Thefluorescence of the solution was used as an accurate measure forpH and hence correlated to the presence of surface carboxyl

Figure 1. Sulfhydryl analyte structures.

4132 Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

groups. The reproducibility of neutravidin coupling was assayedby measuring the fluorescence of nylon mesh materials derivatizedwith FITC-derivatized neutravidin. In this case, the protein-coatedmaterials were placed in wells of a 24-well glass bottom plate andthe fluorescence was measured using a similar set of instrumentconditions. The protein derivatization assay was nondestructiveand thus each batch of materials underwent a quality-controlassessment following this derivatization step.

Execution of Surface-Enhanced TM-DESI Analysis. Theright-hand side of Scheme 1 also provides an overview of theanalytical workflow. First, the pH of the samples was adjusted tothe appropriate range for the capture agent (e.g., ∼10 for VICATSH

capture of sulfhydryls), and a mesh was submerged in eachsample solution to capture the targeted analytes. Analytecapture by the iodoacetaminyl unit of the VICATSH reagent wascomplete in approximately 5-15 min, depending on the volumeof the solution. Next, the meshes were removed from thesamples, rinsed thoroughly with water to remove matrixinterferences (e.g., salts) and nonsulfhydryl-containing com-

pounds, blown dry with air, and placed under a UV lamp for5-10 min to induce photocleavage of the o-nitrobenzyl linkage.The meshes were then placed in line with the electrospray andanalyzed directly by TM-DESI-MS using methanol as theelectrospray solvent. As an example, the photocleavage of asulfhydryl analyte captured by the VICATSH-modified mesh isdepicted in Scheme 2. It should be noted that the current studywas not exclusively aimed at optimizing the quantitative aspectsof the analysis but instead was focused on developing thetechnique for capture, release, and analysis of sulfhydryl analytesfor screening applications. Thus, for more quantitative purposes,a disulfide reduction step utilizing tris(2-carboxyethyl)phosphine

Scheme 1. Workflow for the Manufacture of Surface-Enhanced Materials and the Capture and Analysis ofSulfhydryl Compounds Using Surface Enhanced TM-DESI-MS

Figure 2. Schematic view of surface-enhanced transmission modedesorption electrospray ionization employing VICATSH as a thiolcapture reagent attached to a neutravidin-coated mesh.

Scheme 2. Schematic Overview of the PhotocleavageReaction for a Sulfhydryl-Containing Analyte Capturedby VICATSH

a

a The biotinylated linker molecule remains attached to the meshsurface while the mass tagged analyte is released. The initial photo-cleavage product contains a carbamic acid that undergoes spontaneousdecarboxylation prior to ionization to give the final photocleavageproduct (shown in protonated form). Capture and release results in amass shift of 129 Da of the analyte.

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(TCEP) or another suitable reducing agent should be insertedprior to immersing the mesh materials in the sample in order toensure conversion of nonreactive disulfides to reactive, capturablesulfhydryls.40,41

RESULTS AND DISCUSSIONDESI is an inherently fast ambient ionization mass spectro-

metric technique that may suffer from suppression of lowabundance and low polarity analytes when highly polar interfer-ences are abundant in the sample matrix. However, integrationof DESI with specifically designed chemoselective capture sur-faces can overcome these matrix interferences and also providetargeted analyte enrichment, thereby resulting in improvementsin analytical specificity and sensitivity. The present study detailsthe development of mesh materials specifically designed forsulfhydryl analyte capture, example quality control techniquesuseful in their preparation, optimization of the capture and analysismethods, and application of surface-enhanced TM-DESI for theanalysis of sulfhydryl analytes in complex biological matrices suchas urine and plasma. Specific studies demonstrate the utility andperformance limits of the technique, while simultaneously high-lighting several nuances associated with integrating analytecapture and direct mass spectrometric analysis.

Mesh Manufacture and Quality Control. The developmentof chemoselective and affinity capture techniques is intrinsicallydependent on the number and density of the reactive sites usedto tether capture agents to the surface. Due to incompletepolymerization, some reactive carboxyl and primary amino ter-minal groups are present in most polyamides. However, thenumber and density of these groups can be dramatically increasedby partially hydrolyzing the material, thereby cleaving surfaceamide bonds and creating reactive carboxyl and amino groups intheir place. While polyamide hydrolysis can be accomplishedunder both acidic and basic conditions,44-46 the studies reportedhere utilized an acid-catalyzed hydrolysis with 3 N HCl. Thisconcentration was reported to be optimal for several forms ofnylon44,45 and found here to be the highest tolerable concentrationthe 125 µm mesh strands could withstand. Increasing theconcentration beyond 3 N either dissolved the material completelyor resulted in a severe loss of structural integrity. While exposureof polyamide mesh materials to 3 N HCl undoubtedly resulted inthe creation of additional carboxyl groups, it was also necessaryto evaluate the performance of the hydrolysis reaction.

It is well-known that the fluorescence intensity of an aqueousfluorescein solution is dependent on the equilibrium among itscationic, neutral, anionic, and dianionic forms.47 In the case offluorescein, the dianion and anion have much larger extinctioncoefficients and quantum yields than the neutral molecule.47 Asthe solution pH decreases with the presence of additional carboxylgroups, the shift of the equilibrium from primarily anions anddianions under neutral conditions to fewer anions and neutralspecies results in a large decrease in solution fluorescence. Thus,

an assay that utilized the change in fluorescence of aqueoussolutions of fluorescein following exposure to hydrolyzed meshmaterials was developed as a fast and accurate way to study theefficiency and reproducibility of the hydrolysis, which ultimatelyprovides an indication of mesh carboxylic acid content. Meshsamples were hydrolyzed for durations ranging from 5 min to 24 h.Overall, the extent of hydrolysis was observed to increase withexposure time and eventually plateau after 16-24 h. Furthermore,the reproducibility of the hydrolysis after this extended time periodwas excellent with less than 5% relative standard deviation.

Neutravidin Coupling. In the present study, neutravidin wascoupled directly to the exposed carboxyl groups of the hydrolyzedmesh via a two-step carbodiimide-mediated method to form abinding layer for the subsequent attachment of the biotinylatedphotocleavable reactive capture agent, VICATSH. Because theperformance of surface-enhanced TM-DESI is ultimately de-pendent on the total number of accessible reactive captureagent sites on the mesh, it is critical that the neutravidin surfacelayer used to anchor the VICATSH agent be uniform, robust,and reproducible. To evaluate the preparation of the neutravi-din-modified meshes prior to further derivatization with thebiotinylated reactive capture agent, the performance of theneutravidin-coupling method was assessed by fluorescencemicroscopy and fluorometry using FITC-labeled neutravidin.A fluorescent micrograph of a polyamide mesh successfullyderivatized with FITC-labeled neutravidin alongside a controlmesh that was cleaned and hydrolyzed, but not derivatized withthe protein, is provided in the Supporting Information (FigureS1). The bright green fluorescence observed for the derivatizedmesh compared to the dull outline observed for the control meshprovides convincing evidence that the neutravidin coupling wasboth successful and relatively uniform.

In addition to the qualitative microscopy results, the reproduc-ibility of the neutravidin coupling to the mesh was determinedby quantifying the fluorescence of the meshes. In this case, thematerials were removed from the reaction vials, rinsed with water,blown dry with air, weighed, and assayed for fluorescence. Overall,the derivatization process was quite reproducible as the percentrelative standard deviation over several batches was typically lessthan 10%. Perhaps more importantly, the nondestructive natureof the fluorescence assay facilitated a rapid quality control measurewhich enabled the confident identification and removal of anypoorly performing mesh substrates before they were used forsample analysis.

Coupling of VICATSH. The efficacy of the biotin-neutravidinbinding, and thus the attachment of VICATSH to the neutravidin-derivatized mesh, was assayed by TM-DESI-MS using VICATSH

as a probe molecule in the absence of any sulfhydryl analytes.In this case, representative samples from each preparationbatch were removed from the final derivatization solution,rinsed with water, blown dry with air, and placed under a UVlamp for 10 min to induce photocleavage of the bound VICATSH.The meshes were then subjected to TM-DESI-MS to detectthe release of 4-iodoacetamidyl-butylamine (IABA) from thephotocleaved VICATSH by selected reaction monitoring forconversion of the IABA precursor ion of m/z 257 to the production of m/z 240 upon CID. This product ion was then isolatedand subjected to further CID to produce the MS3 product ions

(44) Edelman, G. M.; Rutishauser, U.; Millette, C. F. Proc. Natl. Acad. Sci. U.S.A.1971, 68, 2153–2157.

(45) Isgrove, F. H.; Williams, R. J. H.; Niven, G. W.; Andrews, A. T. EnzymeMicrob. Technol. 2001, 28, 225–232.

(46) Tang, J.; He, N.; Nie, L.; Xiao, P.; Chen, H. Surf. Sci. 2004, 550, 26–34.(47) Sojback, R.; Nygren, J.; Kubista, M. Spectrochim. Acta Part A 1995, 51,

L7–L21.

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of m/z 198 and 113. A representative extracted ion chronogramfor m/z 198 is shown in Figure 3, while mass tandem massspectra and fragmentation pathways for IABA are provided in theSupporting Information (Figure S2).

The extracted ion chronograms generally showed a distributionof VICATSH across the mesh surface, indicating that the biotinbinding sites are fairly evenly distributed. However, in somecases, larger responses were observed near the edges of themesh. To investigate these anomalies, mesh samples werescanned multiple times across the same path, and while theintensity of the extracted ion chronogram decreased with eachpass, the shape was relatively consistent (i.e., with enhancedsignal intensities near the mesh edge). There are severalpossible causes for these edge effects: (1) they may be relatedto the analysis method (i.e., there is a difference in the mannerin which the electrospray interacts with the mesh at the edges),(2) the mesh materials had more reactive carboxyl groups onthe edges due to more complete hydrolysis of the cut strandcross-section, (3) the materials had more neutravidin on theedges of the mesh due to increased efficiency or nonspecificbinding on the cut edges, or (4) there was increased biotinbinding efficiency at the edges. Further studies are necessarybefore a definitive conclusion can be drawn. In any case, theenhancement of signal near the edges of the mesh did notsignificantly alter the capture and release of the targetedsulfhydryl analytes.

Optimization of Analysis Methods and Capture Condi-tions. As depicted in Scheme 2, photocleavage of VICATSH resultsin the introduction of an easily ionized amine-terminated masstag to each sulfhydryl compound. In the gas phase, eachresulting protonated species undergoes characteristic dissocia-tion pathways upon collisional activation, thus allowing readyidentification by tandem mass spectrometry (MSn). Initialexperiments to establish the fragmentation patterns of VICATSH-tagged analytes were conducted in matrix-free aqueous solu-

tions (100 µM) of each of the five target analytes. The precursorand product ions for each stage of mass spectrometry aresummarized in Table 1. The associated mass spectra and thespecific conditions used for isolation and collisionally induceddissociation of the target analytes are presented in the SupportingInformation (Figure S3-S7).

A series of experiments were performed to assess the impactof the sample pH (7-11), sample exposure time, and photocleav-age conditions on the efficacy of the capture and analysis. Resultsindicated that pH had a tremendous impact on the reaction aslittle to no reaction between VICATSH and the sulfhydryl analyteswas observed at pH values between 7 and 8, a result consistentwith the low nucleophilicity of peptidyl -SH groups versus-S-.48 However, when the pH exceeded 9, the reaction wasvery efficient and no unreacted VICATSH was observed duringthe subsequent ESI-MS analyses. With respect to the captureefficiency and sample exposure time, the capture rate isdependent on the analyte concentration, which affects thefrequency with which analyte molecules interact with the mesh.Thus, it is expected that longer sample exposure times maybe required for extremely low analyte concentrations.

The o-nitrobenzyl group incorporated in VICATSH has beenused in a number of photolabile probes and cross-linkingagents, some of which have become staples of oligonucleotidesynthesis schemes.49-51 Previous reports concerning the use ofVICATSH for quantifying absolute amounts of proteins in celllysates utilized photocleavage times of 16 min,43 while otherreports discussing similar o-nitrobenzyl photolabile compoundsused exposure times as low as 5 min to induce photocleavage.49

Since photocleavage is dependent on the wattage and flux ofthe UV source (in this case the UV lamp was a 20 W lampwith a wavelength of 365 nm and the sample was placedapproximately 5 cm from the lamp), the impact of photocleav-age time on the observed response was also investigated. Inthis study, VICATSH meshes that had not been exposed tosulfhydryl analytes were exposed to UV light for times ranging

(48) Nelson, K. J.; Day, A. E.; Zeng, B.-B.; King, S. B.; Poole, L. B. Anal. Biochem.2008, 375, 187–195.

(49) Olejnik, J.; Sonar, S.; Krzymanska-Olejnik, E.; Rothschild, K. J. Proc. Natl.Acad. Sci. 1995, 92, 7590–7594.

(50) Bai, X.; Li, Z.; Jockusch, S.; Turro, N. J.; Ju, J. Proc. Natl. Acad. Sci. 2003,100 (2), 409–413.

(51) Olejnik, J.; Ludemann, H.-C.; Krzymanska-Olejnik, E.; Berkenkamp, S.;Hillenkamp, F.; Rothschild, K. J. Nucleic Acids Res. 1999, 27, 4626–4631.

Figure 3. The distribution of VICATSH across the derivatized meshwas monitored by TM-DESI-MS. The 1 cm square mesh was affixedto the backing plate and scanned at a rate of 250 µm/s, resulting inexposure of the mesh to the DESI spray for approximately 0.66 min.Photocleavage of unreacted VICATSH followed by desorption elec-trospray ionization and spontaneous loss of CO2 produces the stableion of m/z 257, which corresponds to protonated IABA. Collisionalinduced dissociation (MS3) results in the formation of the product ionof m/z 198 (XIC shown).

Table 1. Precursor and Product Ions for Tandem MassSpectrometry

product m/z

analyte(precursor m/z)a MS2 b MS3 c

mercaptopurine (281) 193, 263, 89 165captopril (346) 231, 249, 213, 329 213, 145, 72penicillamine (278) 89, 260, 217 243, 189, 215, 145acetylcysteine (292) 275, 187, 232, 163, 146, 250 146, 257, 233, 198cysteamine (206) 89, 72, 188 171, 117, 72VICATSH (257) 240 198, 113

a Including the acetamidylbutylamine mass tag transferred from theVICATSH capture reagent. b CID product ions (MS2) are listed in orderof descending abundance. The ion isolated for MS3 analysis is in boldtype. c CID product ions (MS3) are listed in order of descendingabundance.

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between 5 and 30 min, and the detection of IABA (the reporterion from photocleavage of VICATSH) by tandem MS was usedto monitor performance. The results showed that photocleavagetimes of approximately 8-10 min were sufficient using theaforementioned UV source and that longer exposure timesproduced little to no additional response.

Capture of Sulfhydryl Analytes from Biological Matrices.Sulfhydryl compounds are prevalent in pharmaceuticals andhuman metabolism.39-41 Captopril, acetylcysteine, penicillamine,and mercaptopurine are all well-established drugs that are usedto treat a variety of diseases ranging from hypertension tochildhood leukemia.39 Typical doses of these drugs are in therange of milligrams of drug per kilogram of body weight, andconcentrations in plasma are typically in the low micromolar rangeprior to excretion of the excess dose in the urine.52-54 Othersulfhydryl compounds such as cysteine, homocysteine, glu-tathione, and cysteinylglycine are meaningful components of thehuman metabolome, with normal concentrations in the low to midmicromolar range.55 Therefore, it is not surprising that significantefforts have been made to analyze sulfhydryl compounds inbiological matrices, most of which have utilized LC-MS orGC-MS following solution phase derivatization.55-59 The fivecompounds shown in Figure 1 are commonly studied sulfhydrylanalytes that were chosen as a model set to illustrate theapplication of surface-enhanced TM-DESI-MS to biological ma-trices such as urine and plasma. While these analytes are primarilypharmaceuticals, the surface-enhanced technique described herecould be extended to endogenous metabolites, other xenobiotics,or environmental contaminants containing the reactive thiol.

Prior to investigating analyte capture via the surface-enhancedTM-DESI-MS approach, a series of control experiments utilizingblank urine and plasma were performed to probe these matricesfor inherent interferences that could cause false positive identifica-tions of the five target analytes. The results of these controlexperiments indicated that the tandem mass spectrometry analysis(MS3) essentially eliminated false positive analyte identificationsas no target analyte response was consistently observed in anyof the blanks tested. Additional control samples utilizing urineand plasma spiked with captopril were also prepared. In theseanalyses, the mesh substrate was either an underivatized nylonmesh substrate, a mesh derivatized with neutravidin but notwith VICATSH, or a mesh derivatized with both neutravidin andVICATSH but not subjected to photocleavage. In all cases, theresulting mass spectra showed no evidence of captopril or itsVICATSH derivative, thereby confirming that underivatizednylon or neutravidin does not capture sulfhydryl analytes andthat if VICATSH does capture sulfhydryl analytes, then subse-quent photocleavage is essential to produce the derivatizedanalyte spectra.

For comparison, positive control samples were prepared byspiking each of the target analytes into aliquots of either urine orplasma. In these cases, the capture and analysis protocol detailedin Scheme 1 produced unequivocal analyte identification. Forexample, Figure 4 depicts the results of a surface-enhanced TM-DESI-MS analysis of a 1 mL plasma sample containing 50 µMmercaptopurine. The extracted ion chronogram of the CIDproduct ion of m/z 193 obtained upon MS3 (281 f 193 f)confirms that mercaptopurine was successfully captured andretained by the mesh material. In this case, the protonatedspecies of m/z 281 underwent CID by cleavage of the amidebond to produce the ion of m/z 193. Subsequent isolation ofthis product ion followed by a second stage of collisionalactivation favors the production of the ion of m/z 165, whichis indicative of the captured mercaptopurine. Extension of thesepositive control studies to samples with decreasing amountsof mercaptopurine resulted in a limit of detection of ap-proximately 10 nmol, as confirmed by MS3. This limit wasslightly lower using MS2 or full scan analysis, but the benefitof the increased selectivity afforded by the additional stage of

(52) Umrethia, M.; Ghosh, P. K.; Majithya, R.; Murthy, R. S. R. Cancer Invest.2007, 25, 117–123.

(53) Longo, A.; Di Toro, M.; Galimberti, C.; Carenzi, A. J. Chromatogr. 1991,562 (1-2), 639–64.

(54) Richer, C.; Giroux, B.; Plouin, P. F.; Maarek, B.; Giudicelli, J. F. Br. J. Clin.Pharmacol. 1984, 17, 243–250.

(55) Seiwert, B.; Karst, U. Anal. Chem. 2007, 79, 7131–7138.(56) Liang, S. C.; Wang, H.; Zhang, Z. M.; Zhang, H.-S. Anal. Bioanal. Chem.

2005, 381, 1095–1100.(57) Kumierek, K.; Glowacki, R.; Bald, E. Anal. Bioanal. Chem. 2006, 385, 855–

860.(58) Inoue, T.; Kirchhoff, J. R. Anal. Chem. 2002, 74 (6), 1349–1354.(59) Zacharisa, C. K.; Tzanavaras, P. D.; Themelisa, D. G. J. Pharm. Biomed.

Anal. 2009, 50, 384–391.

Figure 4. Surface-enhanced TM-DESI analysis of a 1 mL plasma sample containing mercaptopurine (50 µM). The extracted ion chronogramof the collisionally induced dissociation product ion of m/z 193 is shown on the left, while MS2 and MS3 spectra for the captured analyte areshown on the right.

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mass spectrometry was lost in these cases. Furthermore,incorporation of a disulfide cleavage step in the samplepreparation and improvements to capture agent density on themesh substrate should also result in additional improvementsto the limit of detection.

Figure 5 provides further evidence for sulfhydryl capture, inthis case from urine. A 40 mL urine sample containing penicil-lamine (20 µM) was simultaneously sampled in parallel by usingtwo VICATSH-modified meshes. The extracted ion chronogramfor the CID product ion of m/z 217 (associated with the lossof CO2 and ammonia from the protonated precursor) showsthe successful capture of penicillamine by each mesh. Isolationof the product ion of m/z 217 followed by a second stage ofcollisional activation results in formation of MS3 product ionsof m/z 146, 161, and 200, which confirm the identity of thecaptured penicillamine. Furthermore, this example illustratesthat the sample volume for the surface-enhanced TM-DESI-MS method can be extended to much larger sample sizes (i.e.,40 mL) and a single sample can be exposed to multiple meshesfor replicate analysis or presumably to capture different classesof compounds depending on the nature of the modified mesh.

To further explore the range of sample volumes, 100 µLsamples of plasma were also spiked with mercaptopurine (50 µM).In this case the entire sample volume (100 µL) was deposited ontothe mesh and an additional 20 µL of 0.1 M aqueous sodiumhydroxide was added to the mesh to increase the pH and promotethe capture reaction. The resulting tandem mass spectra andextracted ion chronograms for these experiments resembled thoseobtained for the larger sample volume (Figure 4). Thus, it isconceivable that small plasma or blood spots could be depositedonto mesh substrates, dried, and later reconstituted with anaqueous base solution that effectively dissolved the analytes andfacilitated capture of any free sulfhydryls.

Impact of Mesh Rinsing Method. One common advantageof many capture approaches is the capability to rinse away thesample matrix prior to analysis. In the present case, the covalentcapture of the target analytes was expected to result in a robustmethod that could tolerate thorough rinsing of the capture surface

and thereby overcome the presence of salts and other easilyionized molecules that cause suppression of low-abundancespecies. Therefore, experiments were conducted to investigatethe impact of rinsing the mesh with various solvents (water,ethanol, methanol, acetonitrile) prior to photocleavage. In the firstseries of experiments, VICATSH- derivatized mesh materials wereimmersed in blank urine and plasma. After 10 min of exposure,the mesh materials were removed from the sample and eitherimmediately dried (without rinsing) or rinsed with one of theaforementioned solvents before being subjected to UV photo-cleavage. Again, the performance was measured using tandemmass spectrometry (MS3) of IABA, the photocleavage productof VICATSH.

As shown in Figure 6, sample rinsing was found to greatlyimprove mesh performance. In this example, one of the modified

Figure 5. Surface-enhanced TM-DESI analysis of a 40 mL urine sample containing penicillamine (20 µM). The extracted ion chronogram ofthe collisionally induced dissociation product ion of m/z 217 is shown on the left, while MS2 and MS3 spectra for the captured sulfhydryl analyteare shown on the right. In this case, two mesh materials were submerged in the urine sample and analyzed as duplicates.

Figure 6. Analysis of VICATSH-modified mesh substrates submergedin urine samples. Mesh 1 was rinsed with H2O prior to TM-DESI-MSanalysis, while mesh 2 was air-dried without any additional rinsing.The extracted ion chronogram for the ion of m/z 198 (blue) illustratesthat the response for VICATSH is dramatically reduced when thesample was not rinsed. The extracted ion chronogram for the ion ofm/z 86 (gold) corresponds to the presence of creatinine, an abundantcomponent of urine whose presence causes ion suppression of lowerabundance analytes. Together the two chronograms illustrate theimpact of effective matrix removal following analyte capture.

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meshes (mesh 1) was rinsed thoroughly with water prior to TM-DESI-MS analysis, while a second mesh (mesh 2) was air-driedwithout any additional rinsing. The extracted ion chronogram form/z 198 (depicted in blue) shows strong signals for IABA frommesh 1 but much lower responses for mesh 2, thereby illustratingthat the response for the photocleaved ion was dramaticallyreduced when the mesh was not rinsed. The overlaid extractedion chronogram for m/z 86 (depicted in gold) corresponds to thepresence of creatinine, an abundant component of urine (typicallyin the range ∼1 mg/mL) that is readily ionized due to thepresence of three highly basic nitrogen atoms.60 Without rinsing,the overwhelming abundance of creatinine in the urine clearlycaused suppression of the lower abundance IABA ions.

Experiments conducted using plasma samples produced simi-lar results, again illustrating that rinsing of mesh materials withwater readily removed matrix interferences without noticeabledifferences in IABA response. In contrast, mesh materials thatwere immersed in plasma and subsequently analyzed withoutrinsing showed poor performance due to the viscosity of theresidual plasma and abundance of ionizable plasma components.

A side-by-side comparison for the capture of mercaptopurinefrom water, urine, and plasma was also performed. In this case,an equal amount of mercaptopurine (1 µmol) was spiked into 1mL samples of each matrix. Since the goal of this particular studywas to determine whether rinsing procedures improved methodperformance, the relatively high concentrations of mercaptopurine(1 mM) compared to the natural abundance of endogenous thiolssuch as glutathione, cysteine, and cysteine-glycine ensured thatthe capture of mercaptopurine was stoichiometrically favored overthe other endogenous sulfhydryls. Each spiked sample was thenprocessed using the protocol presented in Scheme 1 and the totalresponse for mercaptopurine was measured by the area of thepeak in the extracted ion chronogram of m/z 193. Resultsindicated that the responses was nearly the same in each case,thereby illustrating that analyte capture followed by mesh rinsingeffectively removes nonsulfhydryl matrix interferences. Similarresults have been previously reported for the surface-enhancedTM-DESI-MS analysis of captopril in the presence of nicotine andhigh salt concentrations.38

Mesh Capacity and Analyte Enrichment. Another commonadvantage of capture methods is the capability of extracting targetanalytes from dilute solutions and thus affording significant analyteenrichment. These methods are similar to other solid-phaseextraction methods in that they provide a means of localizingdissolved analytes to a small surface area; however, in this casethe localization comes with the added benefit of increasedselectivity. The magnitude of the enrichment depends on thedesign of the capture surface and is highly dependent on thesurface area of the material and the density of the capture agents.Experiments were conducted to investigate the ability of the meshmaterials to capture a constant amount of analyte (250 nmol ofcaptopril) in increasingly large sample volumes (1, 5, 50, and 250mL). These solutions ranged from 4 to 250 µM and effectivelytested the ability of the mesh to extract and concentrate thesample. Each spiked sample was processed using the protocol

presented in Scheme 1 (except the capture time was extended to30 min), and the total response for captopril was measured bythe area of the peak in the extracted ion chronogram of m/z 213.Results indicated that captopril could indeed be extracted fromeach sample and that the total area in the extracted ion chrono-gram was fairly consistent. While a net decrease in response ofapproximately 20% was observed for the 250 mL sample, it isreasonable to assume that the recovery could be increased withadditional capture time.

Along with analyte enrichment, all capture methods inherentlyreach a capacity after which no additional analyte can be retained.Again, this property is dependent on the surface area of thematerial and the density of the capture agents on the surface. Totest the capacity of the mesh substrates used in this study,experiments were also conducted wherein a constant samplevolume (5 mL) was spiked with increasing amounts of captopril(0.25, 1, 5, 20 µmol) to create a series of solutions ranging from50 to 4 mM. In these experiments each sample mesh was scannedtwice, once for the presence of captured captopril and once forthe presence of unbound VICATSH, as measured by the detectionof IABA. These results followed the expected inverse relation-ship of analyte abundance (see Supporting Information, FigureS8). As the amount of captopril in solution increases, the amountof captopril captured by VICATSH increases, and thus theobserved response for captopril also increases. Simultaneously,increases in captopril concentration resulted in more captoprilcapture by VICATSH and corresponding reductions in theabundance of free VICATSH. Ultimately, the response forcaptopril appears to plateau at analyte loading amounts around20 µmol, thereby indicating that the mesh has approachedcapacity. The corresponding decrease in the detection of IABAas the amount of captopril increases provides additionalevidence that the majority of VICATSH bound to the surfacewas reacted.

Simultaneous Capture of Multiple Analytes in Urine. Theprevious experiments investigated the capture of a single sulfhy-dryl analyte in a variety of matrices, but they did not directlyaddress the competition between multiple sulfhydryl analytes inthe same solution. Therefore, several experiments were conductedto investigate the performance of the modified meshes when twosulfhydryl analytes (mercaptopurine and captopril) were presentat varying amounts and at various ratios. The results of two ofthese experiments are presented in Figure 7.

In the first example (Figure 7a), 15 µmol of captopril and 7.5µmol of mercaptopurine were spiked into a 40 mL urine sample,thereby resulting in an analyte ratio of 2:1 (captopril:mercaptopu-rine) and a total sulfhydryl amount of 22.5 µmol (neglecting anyendogenous thiols). The results illustrate a few key points: (1)both captopril and mercaptopurine could be captured and identi-fied; (2) the response for mercaptopurine was larger than forcaptopril, even though twice as much captopril was present,potentially indicating a more facile capture of mercaptopurine orhigher ionization efficiency of the derivatized analyte; and (3) theresponse for IABA (displayed as VICATSH in Figure 7) wasrelatively low, indicating that a majority of the VICATSH on thesurface had been reacted, a result that was consistent with theprevious experiments that specifically investigated mesh capacity.

(60) Park, E.-K.; Watanabe, T.; Gee, S. J.; Schenker, M. B.; Hammock, B. D. J.Agric. Food Chem. 2008, 56, 333–336.

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In the second example (Figure 7b), 1 µmol of mercaptopurineand 40 µmol of captopril were spiked into 40 mL urine samples,thereby resulting in an analyte ratio of 40:1 (captopril:mercap-topurine) and a total sulfhydryl amount of 41 µmol. These resultsalso illustrate some key points: (1) the responses for the twoanalytes change in accordance with their molar amounts, (2) asulfhydryl analyte of lower concentration can be detected in thepresence of another of higher concentration, and (3) the totalamount of 40 µmol (mostly from captopril) results in near totalconsumption of VICATSH on the mesh. Results for additionalexperiments, in which the ratio of mercaptopurine to captoprilranged from equimolar to 40:1, produced similar conclusions.Likewise, when the total amount of sulfhydryl analyte wasreduced to the nanomole range instead of micromoles, bothanalytes were detected, but the response for IABA was largerthan that of captopril or mercaptopurine. This result echoesthe data presented in Figure 4 for a single analyte.

While the data presented in Figure 7 demonstrate that multipleanalytes with varying concentrations can be analyzed, it isexpected that there is a limit to the tolerable difference inconcentrations. Ultimately, as the ratio of the major componentto the minor component becomes very large, detection of the

minor component will be precluded simply on the basis ofstoichiometric considerations. In this case, the major componentwould become an interference and a more specific capture agentthat was selective against it would have to be utilized. Thisphenomena is not specific to surface-enhanced TM-DESI, butrather a challenge of chemoselective and affinity capture methodsin general.

CONCLUSIONSThe methods and results presented herein illustrate the

enormous potential for coupling chemoselective capture methodsto ambient ionization mass spectrometry. In this case, targetedsulfhydryl analytes were successfully extracted from complexbiological matrices, and analyte enrichment facilitated the rapidscreening of selected targets via tandem mass spectrometry, whichprovided additional selectivity beyond that already provided bythe specific capture agent. The methods used to produce theselective mesh materials (i.e., nylon hydrolysis, carbodiimide-mediated coupling, avidin-biotin binding) are well-documented,and quality-control assays can be easily incorporated as a meansof discarding poorly constructed materials prior to their use.Furthermore, the covalent capture of analytes allows the use ofan ample rinsing step to remove undesired matrix components,resulting in the dramatic reduction of easily ionized interferencesthat tend to suppress low abundance ions in many desorptionelectrospray ionization analyses.

While this methodology specifically focuses on the capture ofsulfhydryl analytes, the surface-enhanced TM-DESI-MS strategyis a tunable approach and should be amenable to the selectivecapture, release, and analysis of other targeted molecules fromcomplex mixtures through the incorporation of an appropriatecapture agent. Moreover, the results presented here were obtainedusing immediately available mesh materials and establishedderivatization methods. Continued research devoted to developingnew mesh materials and increasing electrospray transmission,capture agent density, and reaction yields will result in improvedperformance.

ACKNOWLEDGMENTSupport from NIH (Grants 1RC1AG035713, J.S.B., and DK67869,

M.H.G.) and the Welch Foundation (F1155 to J.S.B.) is gratefullyacknowledged.

SUPPORTING INFORMATION AVAILABLEFigures S1-S8. This material is available free of charge via

the Internet at http://pubs.acs.org.

Received for review January 27, 2010. Accepted April 6,2010.

AC100242B

Figure 7. Overlaid extracted ion chronograms for surface-enhancedTM-DESI analysis of mixtures of captopril and mercaptopurine inurine. The molar ratio of analytes is given as captopril:mercaptopurine.In each case the chronogram corresponds to the primary MS3 ionlisted in Table 1.

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