Nanostructured Indium Tin Oxide Slides for Small-Molecule Profiling

Nov 20, 2014 - Due to their electrical conductivity and optical transparency, slides coated with a thin layer of indium tin oxide (ITO) are the standa...
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Nanostructured Indium Tin Oxide Slides for Small-Molecule Profiling and Imaging Mass Spectrometry of Metabolites by Surface-Assisted Laser Desorption Ionization MS Carlos López de Laorden,†,# Ana Beloqui,†,⊥,# Luis Yate,‡ Javier Calvo,§ Maria Puigivila,∥ Jordi Llop,∥ and Niels-Christian Reichardt*,† †

Glycotechnology Laboratory, ‡Surface Analysis and Fabrication Platform, §Mass Spectrometry Platform, and ∥Radiochemistry Laboratory, CIC biomaGUNE, Paseo Miramon 182, 20009 San Sebastian, Spain S Supporting Information *

ABSTRACT: Due to their electrical conductivity and optical transparency, slides coated with a thin layer of indium tin oxide (ITO) are the standard substrate for protein imaging mass spectrometry on tissue samples by MALDI-TOF MS. We have now studied the rf magnetron sputtering deposition parameters to prepare ITO thin films on glass substrates with the required nanometric surface structure for their use in the matrix-free imaging of metabolites and small-molecule drugs, without affecting the transparency required for classical histology. The custom-made surfaces were characterized by atomic force microscopy, scanning electron microscopy, ellipsometry, UV, and laser desorption ionization MS (LDI-MS) and employed for the LDI-MS-based analysis of glycans and druglike molecules, the quantification of lactose in milk by isotopic dilution, and metabolite imaging on mouse brain tissue samples. hin films made of indium tin oxide (ITO), a transparent conducting oxide, combine a high optical transmittance in the visible range with high electrical conductivity. Its unique optoelectronical properties and the ease of ITO thin film preparation, e.g., by magnetron sputtering, have been exploited in applications ranging from transparent electrodes and antireflective and display coatings to use in photoelectronic devices such as solar cells or organic light-emmitting diodes.1−3 ITOcoated glass slides have also found use for the preparation of lectin and glycan microarrays with multiple read-out options4 or as transparent and conducting sample plates in imaging mass spectrometry of tissue samples by MALDI-TOF MS. Imaging mass spectrometry (IMS) is a label-free histology technique to study the spatial distribution of proteins, metabolites, lipids, or drugs in thin tissue samples in a nontargeted fashion up to a mass limit of 30−40 kDa.5,6 While classic histology by staining with dyes or fluorescently labeled antibodies can only visualize a limited number of analytes in a single experiment, imaging mass spectrometry can measure the intensities of hundreds of analytes simultaneously.5 The sensitivity of MALDI-TOF MS for the imaging of analytes below 600 Da, however, is compromised by interfering matrix signals7 but can be improved by employing alternative matrixfree ionization techniques such as secondary ion mass spectrometry (SIMS), desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI),8 or surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) on nanostructured surfaces. In addition, the resolution in

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MALDI-TOF MS-based imaging can also be affected by the formation of matrix crystals larger than the laser diameter and further compromised by lateral analyte diffusion during matrix application.6 SALDI-MS matrix-free LDI-MS-based tissue imaging has the potential to produce tissue images with higher resolution, higher reproducibility, and higher spatial fidelity than MALDI-TOF MS as lateral metabolite diffusion during matrix application and sweet spot formation through heterogeneous crystallization are avoided. With the development of reproducible and sensitive LDI-active surfaces, label-free imaging of metabolites and drugs on tissue sections by mass spectrometry is likely to gain importance in drug discovery, clinical histology, pharmacokinetics, and basic molecular biology.6,9,10 A large number of materials have been evaluated as substrates for SALDI-MS, including the carbon allotropes grapheme,11 carbon nanotubes,12 and graphite13 or thin films made of semiconductors,14−16 metals,17−19 or metal oxides,17,20 many with unique optical thermal and electric properties that facilitate soft ionization of absorbed analytes under reduced laser power.21 Most applications of LDI-active substrates have focused on enzymatic assays,22 forensics,23 metabolite identification in biofluids,24 or potential platforms for food analysis,20 and only Received: July 14, 2014 Accepted: November 20, 2014

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a few have been used for imaging mass spectrometry.10,25,26 With the exception of a semitransparent nanostructure initiator mass spectrometry (NIMS) surface,27 all reported materials employed in IMS are opaque and are not suitable for complementary histology using optical microscopy. As an alternative to LDI-active surfaces, nanoparticle-based approaches that substitute the organic matrix for a solution of colloidal gold,28 silver,29 or titanium oxide7 nanoparticles or graphite13 have been developed for IMS. Through our previous work on ITO-based glycan microarrays, we were tempted to study ITO thin films also as potential substrates for SALDI-MS and in particular for IMS on standard MALDI-TOF mass spectrometers. Recently, Goodwin et al. employed a 12 T Fourier transfer ion cyclotron resonance (FTICR) mass spectrometer for the matrixless detection of phospholipids from a tissue sample on a commercial ITO slide.30 The sensitivity of conventional MALDI-TOF MS instruments, however, is not sufficient for this matrix-free application. Here we describe the specific preparation and use of ITOcoated glass slides as an inexpensive, transparent, and conductive material for SALDI-MS on standard MALDI-TOF mass spectrometers. ITO slides were custom-made by radio frequency (rf) magnetron sputtering and applied as sample plates in the matrix-free analysis of small molecules and drugs, the quantifaction of lactose in milk samples, and the imaging of the spatial metabolite distribution on mouse brain tissue samples.

NanoWizard II) in intermittent contact tapping mode using a tapping etched silicon probe (TESP) with a 0.01−0.025 Ω/cm antimony (n) doped Si, rectangular, 4 μm thick cantilever with a nominal resonant frequency, spring constant, length, and width of 320 kHz, 42 N/m, 125 μm, and 40 μm, respectively. Images were obtained with a line rate of 0.6 Hz, an initial set point of 500 mV, an integral gain of 40.0 Hz, and a proportional gain of 0.002 with a pixel resolution of 512 × 512. The scanning area was 8 × 8 μm. Topology parameters and AFM images and cross sections are provided in the Supporting Information (Table S-4, Figures S-1 and S-2). The transmission spectra of the ITOcoated glass slides were performed by using a UV Beckman Coulter DU 800 UV/vis/NIR spectrophotometer equipped with a special slide adapter in transmission mode from 200 to 800 nm. The wavelength interval and scan speed were set at 0.2 and 240 nm/min, respectively (Supporting Information, Figure S-3). The thickness and resistivity of the ITO films deposited on a silicon wafer were measured with a rotating spectroscopy ellipsometer (W2000 V J.A Woollam Co., Inc.) at several angles of incidence between 50° and 70°. Analysis of the ellipsometry measurements was carried out using an ITO parametrized function with a model dielectric function having a combination of Lorentz oscillators and the Drude term (Supporting Information, Table S-3). The contact angle of each ITO deposited film was measured in triplicate using a Krüss DSA 100 instrument. The dosing volume was 1 mL/min. Grain Size Calculation. The average grain diameter was calculated using the WSxM 4.0 software package, Nanotec Electronica S.L., Spain. To obtain area values, the Flooding option was used, which determines the number of hits (grains) in an image and the total area they represent at a certain height. By dividing the total detected area by the number of hits, the average grain diameter was obtained. Elemental Composition by X-ray Photoelectron Spectroscopy (XPS). XPS experiments were performed in a SPECS Sage HR 100 spectrometer with a nonmonochromatic X-ray source (Mg Kα line of 1253.6 eV energy and 250 W) and calibrated using the 3d5/2 line of Ag with a full width at halfmaximum (fwhm) of 1.1 eV. The selected resolutions were 30 eV of pass energy and 0.5 eV/step for the general survey spectra and 15 eV of pass energy and 0.15 eV/step for the detailed spectra of the different elements. All measurements were made in an ultrahigh vacuum (UHV) chamber at a pressure below 5 × 10−6 Pa. In the fittings of the O 1s photoelectron peaks, Gaussian− Lorentzian functions were used (after a Shirley background correction) where the fwhm’s of all the peaks were constrained while the peak positions and areas were set free. Mass Spectrometry. Mass spectra were recorded on a Bruker Ultraflextreme III TOF mass spectrometer equipped with a pulsed Nd:YAG laser (λ = 355 nm) and controlled by FlexControl 3.3 software (Bruker Daltonics, Bremen, Germany). The acquisitions (total of 3000) were carried out in positive or negative reflector ion mode with a pulse duration of 50 ns, a frequency of 1000 Hz, a laser fluence of 40−60%, and the following laser focus settings: offset = 0%, range = 100%, and value = 12%. The m/z range was chosen according to the mass of the sample. The accumulated spectra were then treated with FlexAnalysis v3.3 software. For lactose quantification the measurements were performed as follows: 3000 shots in random walk mode per replicate spot, ion source 1 (IS1) voltage of 25.18 kV, ion source 2 (IS2) voltage of 22.33 kV, lens (L) voltage of 7.53 kV, reflector 1 (R1) voltage

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EXPERIMENTAL SECTION Materials. Commercial ITO slides (75 mm × 25 mm) were purchased from Hudson Surface Technology, Inc. (Old Tappan, NJ), Bruker Daltonics (Bremen, Germany), and VisionTek Systems Ltd. (Cheshire, U.K.). Glass slides (3 × 1 in., Corning 2948) employed for ITO coating were purchased from Corning (Corning, NY). (3-Aminopropyl)triethoxysilane (APTES) was purchased from Sigma-Aldrich and used without further purification. Stearyl succinimide ester was prepared as described previously.31 Semiskimmed and lactose-free skimmed milk samples used in the study were purchased from Kaiku Corporación Alimentaria (San Sebastian, Spain) with fat contents of 1.5 and less than 0.3 g/100 mL, respectively. A 13 C12-labeled lactose standard was purchased from Omicron Biochemicals Inc. (South Bend, IN). Indium tin oxide target was purchased from AJA International Inc. (North Scituate, MA). Deposition of ITO Films. ITO films were deposited by an rf magnetron sputtering system (AJA ATC 1800 UHV) on glass substrates. A 2 in., 90% In2O3 and 10% SnO2 by weight (99.99% purity), target was used as the sputtering source. The sputtering chamber was evacuated prior to sputtering to a base pressure of around 10−6 Pa and then backfilled with argon gas. The rotation speed (80 rpm) and deposition temperature (room temperature) were kept constant for all the experiments. The different films were deposited with varying deposition pressure and rf power between 0.4 and 1.1 Pa and between 10 and 60 W, respectively. A detailed description of the sputtering parameters for slides ITO-1 to ITO-12 is given in the Supporting Information, Table S-1. All ITO-4 slides were treated with a diluted basic piranha solution (10 mL of concentrated H2O2 (30%), 10 mL of concentrated NH4OH (25%), and 50 mL of H2O) at 60 °C for 1−2 h, washed with deionized water until the washing solutions were neutral, and left immersed in water until use. Structure and Topology Characterization. The surface topologies of ITO-sputtered slides ITO-1 to ITO-12 were characterized by atomic force microscopy (AFM; JPK B

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metabolite structure) and confirmed with a reported mouse brain IMS study.32 The MS/MS spectra were obtained in negative ionization mode from accumulation of at least 2000 laser shots and using a pulsed ion extraction of 70 ns. The parent mass was selected manually at m/z 888 (monoisotopic peak), and spectra were acquired in the range of m/z 50−900. The following voltages were used for the analysis: acceleration voltages of 7.5 kV for IS1 and 6.85 kV for IS2, reflector voltages of 29.5 kV for R1 and 13.95 for R2, and LIFT voltages of 19 kV and 3.3 kV for L1 and L2, respectively.

of 26.37 kV, reflector 2 (R2) voltage of 13.50 kV, and positive reflector mode in a mass range of 100−600 Da. The laser energy was set to 55 μJ/pulse. LDI-MS images were constructed using FlexImaging 2.1 software with a spatial resolution of 20−70 μm, in both positive and negative reflector ion mode, 500 shots per spectrum, a laser fluence of 55%, and the following laser focus settings: offset = 0%, range = 100%, and value = 85%. Lactose Quantification. The isotopic dilution assay for lactose in milk, employing 13C-labeled lactose as an internal standard, was validated by determining the linear range, reproducibility, limit of detection (LOD; S/N > 3), and limit of quantification (LOQ; S/N > 5) of the method. For the determination of the linear range, LOD, and LOQ, six different concentrations of labeled lactose were prepared (336, 168, 129, 60, 17, and 8.5 mg/L) in a cow milk working solution. The working solution was prepared by 1:10 dilution of the sample (100 μL to 1 mL) with water followed by a second 1:10 dilution with 70% acetonitrile (1:100 final dilution). The final dilution was centrifuged (13000g for 2 min) and the supernatant recovered. These solutions were directly spotted (0.5 μL) in triplicate onto the ITO-coated glass slides. After air-drying, the spots were submitted to LDI analysis. A 1 mM stock of 13C-lactose standard was prepared in 70% acetonitrile. To 10 μL of lactose-free milk were added 25 μL of 1 mM standard and 65 μL of water. A 10 μL volume of this solution was first diluted to 200 μL with 70% acetonitrile, making a 1:200 milk dilution with a 13C-lactose standard concentration of 12.5 μM. Two independent sample preparations were measured in triplicate to examine the reproducibility of the method. The lactose quantification was performed considering the sum of the area under the peaks of sodium and potassium adducts of analyte and internal standard (Supporting Information, Table S-5). Tissue Imaging. A single mouse brain, obtained from the Molecular Imaging Unit, CIC biomaGUNE, was cut with a cryostat microtome (Leica CM-3050S) into 4 and 10 μm thick slices at −20 °C, which were thaw-mounted onto ITO slides. The optimal tissue thickness was found to be 4 μm. All animal studies in this unit are approved by the Animal Ethics Committee of CIC biomaGUNE and conducted in accordance with the Directives of the European Union on animal ethics and welfare. Hydrophobic Coating of ITO Slides. After being washed with basic piranha solution for 1 h at 60 °C (see above), ITO-4 slides were modified subsequently with APTES (2% in dichloromethane (DCM)) for 2 h at room temperature and stearyl succinimide ester in DMF with N,N-diisopropylethylamine as described previously.4 Surface hydrophobicity was assessed by contact angle measurements. After deposition of the tissue slices, the slides were stored overnight in a vacuum chamber. These samples were directly used for LDI-MS experiments. Hydrophobically coated slides were washed three times for 15 s under sonication in water and air-dried before imaging. Metabolite Identification. Metabolites (adenosine diphosphate (ADP), stearic acid, glycerol monophosphate, palmitic acid) were tentatively assigned on the basis of exact mass (m/z) queries using the databases Human Metabolome Project (http://redpoll.pharmacy.ualberta.ca/hmdb/HMDB/) and METLIN (http://metlin.scripps.edu/). The metabolite ion 888 Da was assigned to a sulfated galactocerebroside on the basis of LIFT-based fragmentation (see the Supporting Information, Figure S-5, for MS/MS and the proposed

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RESULTS AND DISCUSSION ITO thin films can be obtained by pulsed laser ablation, by evaporation (thermal or e-beam), or by dc/rf magnetron sputtering the latter producing films with lowest resistivity, highest transmission, and exceptional reproducibility.33−35 The optimization of sputtering parameters to obtain thin films with improved transmission and desired photoelectric properties has been reported.36−38 The control of the sputtering process to produce ITO surfaces with particle dimensions that facilitate soft laser desorption ionization, however, has not been described so far. The interaction of pulsed laser light with a nanostructured surface can induce nonlinear processes, not observed for the bulk material, which increase significantly the efficiency for analyte desorption and ion production in the SALDI process.39 The mechanism for this signal enhancement will depend largely on the substrate material and its surface structure, the irradiating laser wavelength and power, and the adsorbed analyte among other factors.21,39 ITO thin films usually exhibit transmission spectra very similar to that of glass and a generally low absorbance at laser wavelengths typically employed in mass spectrometers. An increase in UV absorption could improve desorption of adsorbed analytes by energy dissipation through rapid substrate heating. Our first goal was therefore to increase absorbance at 355 nm by changing the grain size of ITO surface nanoparticles without sacrificing the high transparency in the visible spectra required for histology by optical microscopy. We prepared a first set of eight ITO substrates (ITO-1 to ITO-8) with a targeted film thickness of around 20−50 nm by varying the sputtering rf power and gas pressure and by adjusting the sputtering time to values between 20 min and 2 h. By increasing the deposition time to values between 3 and 5 h, a second batch of slides specified as ITO-9 to ITO-12 were prepared with a thicker ITO layer between 50 and 290 nm. Table S-2 (Supporting Information) summarizes the detailed deposition parameters employed for the preparation of samples ITO-1 to ITO-12. Figure S-3 (Supporting Information) shows the transmission spectra for all custom-made and commercial slides. While slides ITO-10 to ITO-12 are opaque and unsuitable for optical microscopy, ITO-4, ITO-9, and three commercial slides showed a clearly reduced transmission in favor of enhanced absorption (as reflectance can be largely neglected) in the UV region compared to the untreated glass substrate. The elemental composition, topology, electrical properties, and hydrophobicity of all substrates were determined by XPS, AFM, ellipsometry, and contact angle measurement, respectively (see Table S-3, Supporting Information). SALDI-MS performance was measured as the S/N ratio for the detection of the model analytes 1-aminohexaethylene glycol, leucine enkephalin, and lactose (Table S-1, Supporting Information). As expected, changes in sputtering parameters had a strong effect on the surface hydrophobicity, optical transmission, roughness, resistivity, and performance in SALDI-MS. In general, C

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Figure 1. (A) AFM images of ITO-4 and a commercial ITO slide. (B−D) Correlation of S/N ratios for detection of lactose at 0.04 mg/mL with different surface parameters for conductive ITO slides (nonconductive ITO-3 and ITO-7 not included): (B) influence of transmission at 355 nm on SALDI-MS performance, (C) dependence of SALDI-MS performance on average particle grain size, (D) correlation of surface roughness with the S/N ratio. Key: green triangles, grouped nontransparent ITO substrates ITO-10 to ITO-12 with similar grain size larger than 100 nm; black circles, grouped ITO substrates with similar grain sizes below 50 nm.

a higher surface roughness measured by AFM was obtained at lower rf power and lower argon pressure (lower deposition rate), both favoring crystal growth to larger grain sizes. Addition of oxygen as a reactive gas during sputtering (ITO-3 and ITO-7) resulted in nonconductive surfaces probably due to changes in the structure and/or stoichiometry of the samples. Elemental surface composition analysis by XPS and subsequent fitting of the O 1s peak (data not shown) showed a higher percentage of free hydroxyl functions for slides ITO-4, ITO-5, and ITO-8, an advantage for any further surface functionalization. The smaller contact angle and higher hydrophilicity observed were in agreement with the higher hydroxyl group content of these surfaces. The slides ITO-3 and ITO-7 and all commercial substrates did not produce any signal in SALDI-MS with any tested analyte even at high fluence and increased analyte concentration. The opaque surfaces ITO-10 to ITO-12 with a thicker ITO coating showed good SALDI performance at high concentration for detection of leucin enkephalin and lactose but low performance for the detection of lactose at low concentration. Our criteria for selecting a particular substrate for further investigation were (1) high optical transparency and (2) the best possible SALDI performance at low analyte concentration as a measure of sensitivity. With an exceptional performance at the lowest lactose concentration and good overall S/N at all other tested conditions, the fully transparent slide ITO-4 was selected as the most sensitive substrate for SALDI-MS to assess the scope of the method in small-molecule analysis and tissue imaging. From the correlation of selected surface parameters with S/N ratios (at 0.04 mg/mL lactose concentration), we could observe that the performance of substrate ITO-4 is not due to a single

surface attribute but rather the result of a beneficial combination of surface parameters (Figure 1). A comparison of the surface parameters for the conductive but nonactive commercial substrates with the SALDI-active custommade substrates ITO-1, ITO-2, ITO-4, ITO-5, ITO-6, and ITO9 suggests that the ITO SALDI activity is the consequence of a particular surface roughness of close to 1 nm, a grain size between 50 and 100 nm, and a specific film thickness of 20−40 nm. The reduced transmission observed for several commercial slides together with their complete lack of activity as a substrate for LDI-MS might also be explained by an increase in reflectance rather than adsorption due to the larger particle grain size or amorphous surface structure of these substrates.39,40 Of the substrates which show surface activity for LDI-MS (ITO-1, ITO-2, ITO-4, ITO-5, ITO-6, and ITO-9) albeit with distinct performance, slide ITO-4 can be described as a smooth, nonporous layer of 60 nm large ITO nanoparticles (see Figure 1A) with a thickness of 25−35 nm which shows an improved absorption (30%) at the laser wavelength (Figure 1B). As the grain size and film thickness are known to affect the band gap of semiconductors, their adjustment as shown for indium tin oxide here can improve absorption at a desired wavelength.41 Analyte desorption is likely to occur thermally after rapid local heating of the substrate under laser irradiation. Similar to titanium dioxidecoated surfaces,42 indium tin oxide thin films show a high proton affinity,43 and ion formation will be dominated by cationization in the desorption plume with sodium and potassium which are readily desorbed from the surface and lead to a soft ionization of the analytes. Several batches of ITO-4 slides were prepared by rf sputtering, a process known to be highly reproducible when all parameters D

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Figure 2. Selection of lower weight molecules analyzed by LDI-MS on custom-made ITO plates (ITO-4). Key: F, possible fragment ion or impurity; asterisk, observed ITO-related background ion (see the Supporting Information). All spectra were acquired in positive mode except spectra for dihexadecyl-sn-glycerophospoethanolamine (negative ion mode).

are well controlled,44 and the scope of the SALDI-MS method was evaluated on low-mass analytes, including serum metabolites, carbohydrates, lipids, and peptides deposited at picomole quantities by LDI-MS. Figures 2 and 3 show representative spectra for the compounds creatinine (0.5 μL, 25 pmol, m/z = 136.08 [M + Na]+, 152.06 [M + K]+), taurine (0.5 μL, 250 pmol, m/z = 148.01 [M + Na]+), lamivudine (0.5 μL, 250 pmol, m/z = 252.08, [M + Na]+, 268.06 [M + K]+), laminarin hexaose (0.5 μL, 505 pmol, m/z = 1013.55 [M + Na]+), dihexadecyl-sn-glycerophosphoethanolamine (0.5 μL, 750 pmol, m/z = 662.85 [M − H]−), n-octyl β-thioglucoside (0.5 μL, 170 pmol, m/z = 331.26 [M + Na]+, 347.25 [M + K]+), leucine enkephalin acetate salt hydrate (0.5 μL, 0.1 mg/mL, m/z = 578.41 [M + Na]+, 594.31 [M + K]+, 600.39 [M′ + Na]+, 616.37 [M′ + K]+), hexaethylene glycol (0.5 μL, 25 nmol, m/z = 305.30 [M + Na]+, 321.28 [M + K]+), and the Gly-Cys dipeptide (0.5 μL, 280 pmol, m/z = 201.06 [M + Na]+). Other substrates analyzed on this surface included the lipid fraction of human milk (Sigma, 10% (w/v) solution in chloroform, palmitic acid (m/z = 255.53 [M − H]−), oleic acid (m/z = 281.5 [M − H]−), among other lipids), a synthetic C5-amino-modified N-glycan45 (0.5 μL, 25 pmol, m/z = 1059.99 [M + Na]+, 1076.04 [M + K]+), and immunoglobulin N-glycans enzymatically cleaved by PNGase treatment (fucosylated biantennary N-glycan, m/z = 1486.0; fucosylated monogalactosylated biantennary N-glycan, m/z 1648.2; fucosylated bisgalactosylated biantennary N-glycan, m/z = 1810.4). Most analytes ionized as sodium and potassium adducts, and clean spectra with little or no interference from background ions were usually obtained. For compounds which were less effectively ionized (e.g., taurine, laminarin hexasaccharide, N-glycans), indium tin oxide-related and sodium and potassium background ions and in-source fragmentation could be observed. Reoccurring background ions with significant intensity were

tentatively assigned to indium (m/z 115.05) and indium oxide (In3O; m/z 360.70) species, respectively. We analyzed a dilution series (from 1000 to 0.5 pmol) for creatinine (m/z = 136.04 [M + Na]+) to measure the sensitivity of the LDI detection method. The method produced goodquality spectra down to 0.5 pmol of deposited analyte with a signal-to-noise ratio of 13, comparable to the sensitivity of reported nanostructured gold46 and metal oxide LDI methods,47 although a direct comparison has not been made (Figure 4). Lactose Quantification. Lactose quantification in so-called lactose-poor and lactose-free milk products is an important analytical application in the dairy industry calling for inexpensive and high-throughput methods that can substitute for the timeconsuming chromatographic methods and enzymatic cascade assays.48−52 We have tested the utility of our matrix-free LDI sample plate for the quantification of lactose in milk samples by isotopic dilution employing 13C-labeled lactose as an internal isotopic standard. Five milk samples with varying fat and lactose content were diluted up to 100-fold with water and acetonitrile and their lactose contents quantified by relating the integrated peak area for lactose to the internal standard of known concentration. The dynamic range, limit of detection, and limit of quantification of the method were determined by adding known amounts of isotopically labeled standard to the milk matrix and plotting the peak intensity against the analyte concentration (Figure 5). Lactose in diluted milk samples can be quantified down to 10 mg/L, sufficient for the analysis of lactose-free labeled products (100 mg/L) with high sample speed and no other sample preparation than dilution with water and acetonitrile. The sensitivity of our method is comparable with the reported limit of quantification by LC methods with UV detection (6 mg/L) but avoids extensive sample preparation and sampling times of around 25 min.49 E

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Figure 3. Selection of some higher weight molecules analyzed by LDI-MS on custom-made ITO plates (ITO-4): dotted box, expanded regions of interest (all spectra were acquired in positive mode except spectra of the lipid fraction of human milk (negative ion mode)); F, possible fragment ion or impurity; asterisk, observed ITO-related background ion (see the Supporting Information for a full list of ITO background ions).

Figure 4. LDI-MS sensitivity assay. SALDI-MS spectra of creatinine spotted at 1000, 60, 25, and 0.5 pmol (sodium and potassium adducts).

with APTES and stearylsuccinimide ester31 to build up a hydrophobic layer to facilitate the extraction of lipophilic components from the tissue sample. Although the surface modification with

As a further application we employed sputter-coated ITO slides for imaging mass spectrometry of mouse brain tissue slices. The indium tin oxide surface was subsequently functionalized F

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Figure 5. Lactose quantification in milk. (A) From top to bottom, LDI-MS spectra of lactose, 13C-labeled lactose, milk sample, milk with added 13 C-labeled lactose standard, and lactose-free milk with internal standard. Performance parameters for lactose quantification in milk by isotopic dilution: (B) dynamic range, (C) limit of detection (m/z 377) and limit of quantification (m/z 393).

Frozen mouse brain tissue slices of 4 and 10 μm thickness (in the frozen state) were thaw-mounted onto sample slides, dried, stored overnight under vacuum, and analyzed either directly by SALDI-MS or after removal of the tissue by combined washing and sonication (stamping method).26 Direct SALDI-MS

di- and triethoxyalkylsilanes has been reported to potentially suppress LDI,24 we could observe a general increase in metabolite coverage and sensitivity in both positive and negative modes on the hydrophobically coated ITO slides (Figure S-6, Supporting Information). G

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Figure 6. Spatial distribution of selected metabolite ions in a mouse brain tissue sample at 50 μm resolution without removal of the tissue. Images were acquired in negative ion mode.

Figure 7. Spatial distribution of metabolites scanned at 50 μm resolution by IMS in negative ion mode after removal of the tissue by washing. Several peaks were tentatively assigned to major metabolites: m/z = 171, glycerol monophosphate; m/z = 255, palmitic acid; m/z = 283, stearic acid, m/z = 426, ADP; m/z = 888, sulfated galactocerebroside.

samples by SALDI-MS is thought either to happen after tissue ablation down to the underlying nanostructure by repeated laser shots53 or to be facilitated by UV-light-absorbing metabolites present in the sample and which can act as an internal matrix.54 Prior to SALDI imaging of tissues by the stamping method, the

of the tissue slices produced appreciable analyte signal strength only for the thin 4 μm tissue slices, but the analysis of the hydrophobic surface impregnated with metabolites after tissue removal resulted in spectra with much improved signal intensity. Analyte desorption ionization during direct analysis of tissue H

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Author Contributions

repeated washing and sonication of the ITO slide removed the tissue from the slide without touching metabolites extracted from the biological matrix by the hydrophobic surface. Figures 6 and 7 show the spatial distribution of a selection of metabolites acquired at 40 and 50 μm resolution and in negative ion mode on an axial section of a mouse brain. Even though the axial brain section employed in this proof of concept experiment showed little structural heterogeneity by visual inspection under the microscope, SALDI-MS imaging was able to trace a spatially differentiated distribution or absence of certain metabolites to the corpus callosum (m/z = 131, 173, 281, 888) or the cortex (e.g., m/z = 125, 150). Metabolite distribution in the mouse brain by mass spectrometry has been reported by Setou et al. employing TiO2 nanoparticles as a matrix with good metabolite coverage,7 but this procedure adds a sample preparation step and requires specialized instrumentation for spray-coating the tissue sample with nanoparticles.

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C.L.d.L. and A.B. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge funding from The Spanish Ministry of Economy and Competiveness (MINECO; Grant CTQ201127874), the Government of the Basque Country (Etortek Grants 2013 and 2014), and the Guipuzcoan Regional Government (Guipuzcoan Program of Science, Technology and Innovation). We thank Prof. Manuel Martin-Lomas for helpful discussions and proof-reading of the manuscript.

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CONCLUSIONS We demonstrate that a precise control of sputtering parameters can produce nanostructured indium tin oxide thin films with adequate grain size, film thickness, and absorption profile for applications in small-molecule SALDI-MS. A number of commercially available ITO slides were compared to the custom-made material but found to be unsuitable for SALDI-MS on a standard MALDI-TOF spectrometer. The substrates obtained by our rf magnetron sputtering procedure are conductive, nanostructured, and transparent and show high potential for the matrix-free imaging mass spectrometry of small molecules, conventional matrix-assisted imaging of larger lipids and proteins, and traditional histology by optical microscopy. Nanostructuring of the ITO surface therefore broadens the applications of ITO slides in IMS to include matrix-free smallmolecule imaging, without compromising transparency or performance in matrix-assisted imaging. Other applications include the general matrix-free analysis of small molecules, metabolites, and drugs on standard MALDI-TOF mass spectrometers at a fraction of the time and price of ESI-TOF spectrometers working in the solution phase. The production of ITO slides by rf magnetron sputtering is highly reproducible and results in slides with high surface homogeneity, important parameters for future applications in surface-based mass spectrometry. Similar to that on many other surfaces developed for SALDI-MS, the analysis on nanostructured ITO slides is still limited to a mass range of below 2 kDa. Further functionalization of the ITO substrates with capture ligands or enzyme substrates will permit the development of array-based high-throughput assays for applications in diagnostics and drug discovery.

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ASSOCIATED CONTENT

S Supporting Information *

Surface characterization data, including AFM micrographs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ⊥

A.B.: Institute for Chemical Technology and Polymer Chemistry, KIT, Engesserstrasse 18, 76131 Karlsruhe, Germany. I

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