Nanostructured Diamond-Like Carbon on Digital Versatile Disc as a

Anal. Chem. 2008, 80, 7467–7472

Nanostructured Diamond-Like Carbon on Digital Versatile Disc as a Matrix-Free Target for Laser Desorption/Ionization Mass Spectrometry Muhammad Najam-ul-Haq,†,‡ Matthias Rainer,† Christian W. Huck,† Peter Hausberger,§ Harald Kraushaar,§ and Gu¨nther K. Bonn*,† Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020 Innsbruck, Austria, Department of Chemistry, Bahauddin Zakariya University, 60800 Multan, Pakistan, and Sony DADC AG, Sonystrasse 20, A-5081 Anif, Austria A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrixfree sample support for application in laser desorption/ ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp2 carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/µL for [Glu1]-fibrinopeptide B (m/z 1570.6) and 1 fmol/µL for L-sorbose (Na+ adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device. Carbon nanomaterial research has strong implications in metabolomics, peptidomics, and proteomics based on laser desorption/ionization mass spectrometry (LDI-MS). The size that draws most attention for such fields ranges from 100 to 0.2 nm and the bulk characteristics are enormously different, when one moves to the nanostructures. Nanostructured materials have a * Corresponding author. Gu ¨ nther K. Bonn, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria, Europe. Phone: +43 512 507 5171. Fax: +43512 507 2943. E-mail: [email protected]. † Leopold-Franzens University. ‡ Bahauddin Zakariya University. § Sony DADC AG. 10.1021/ac801190e CCC: $40.75  2008 American Chemical Society Published on Web 08/27/2008

recent history in LDI-MS.1-3 In 1999, matrix-free desorption/ ionization on porous silicon (DIOS) was introduced for analyzing low-molecular mass compounds.4,5 DIOS surfaces are usually manufactured by galvanostatic etching procedures and show optimal performance for molecules less than 3000 Da.6,7 It is speculated that silicon films are capable of trapping analytes in its pores and simultaneously absorbing laser’s radiation leading to spectra without matrix background. Nevertheless, one should consider the limits of DIOS-surfaces regarding handling and storage.8 Porous silicon substrates are shown to be sensitive to contamination and shelf life is limited to 1 year after production even by keeping them under special storage conditions.9 The reduced durability is also a problem that can be observed by using sol-gel-assisted substrates. This approach, termed as sol-gelassisted laser desorption/ionization (SGALDI) mass spectrometry uses organic/inorganic hybrid films as sample supports to generate mass spectra without suppressing the analyte’s signals or forming clusters. Moreover these silicon films generated by plasma-enhanced chemical vapor deposition can be successfully applied for the analysis of small organics.10 The lifetime of the most commonly used SGALDI-supports does not exceed more than 1 week, as presumably condensation effects harm the surface, resulting in a decreased signal intensity.8 Other silicon based matrix-free materials involve DIOM (desorption/ionization on mesoporous silicate)11 and the silicon dioxide chip, termed as (1) Chen, C. T.; Chen, Y. C. Rapid Commun. Mass Spectrom. 2004, 18, 1956– 1964. (2) Guo, Z.; Ganawi, A. A. A.; Liu, Q.; He, L. Anal. Bioanal. Chem. 2006, 384, 584–592. (3) McLean, J. A.; Stumpo, K. A.; Russell, D. H. J. Am. Chem. Soc. 2005, 127, 5304–5305. (4) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243–246. (5) Budimir, N.; Blais, J. C.; Fournier, F.; Tabet, J. C. Rapid Commun. Mass Spectrom. 2006, 20, 680–684. (6) Thomas, J. J.; Shen, Z.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4932–4937. (7) Lewis, W. G.; Shen, Z.; Finn, M. G.; Siuzdak, G. Int. J. Mass Spectrom. 2003, 226, 107–116. (8) Dattelbaum, A. M.; Iyer, S. Expert Rev. Proteomics 2006, 3, 153–161. (9) Credo, G.; Hewitson, H.; Benevides, C.; Bouvier, E. S. P. Mater. Res. Soc. Symp. Proc. 2004, 808, 471–476. (10) Lin, Y. S.; Chen, Y. C. Anal. Chem. 2002, 74, 5793–5798. (11) Lee, C. S.; Lee, J. H.; Kang, K. K.; Song, H. M.; Kim, I. H.; Rhee, H. K.; Kim, B. G. Biotechnol. Bioprocess. Eng. 2007, 12, 174–179.

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DIOSD (desorption/ionization on silicon dioxide).12 Alternative sample supports for the use in matrix-free laser desorption/ ionization (LDI) are carbon materials like graphite,13 carbonnanotubes,14 fullerenes,15 or amorphous carbon.16 Graphite plates have been used for measuring polymeric substances in the range from m/z 100-1000 by LDI-MS.17 Moreover they are employed in visible surface-assisted desorption/ionization mass spectrometry (SALDI-MS) for the detection of small macromolecules like synthetic polymers and biomolecules, with a 532 nm laser.18 It is argued that visible-SALDI has a softer ionization procedure in comparison to UV-MALDI. Still some improvements have to be made in the case of fullerenes and fullerene-derivatives as they show poor sensitivity as an effective MALDI-matrix.19 In contrast to fullerenes, carbon-nanotubes show very high sensitivity but are not easily soluble in aqueous solutions, which limit their use in bioanalytical applications. In recent times, the hydrophobic porous surfaces of monoliths like poly(butyl methacrylate-co-ethylene dimethacrylate), poly(benzyl methacrylate-co-ethylene dimethacrylate), and poly(styrene-co-divinylbenzene) have been employed for the mass determination of drugs, explosives, and acid labile compounds.20 Polymer-assisted laser desorption/ionization (PALDI) matrixes21 based on oligothiophene or a benzodioxin backbone are employed in the analysis of low-molecular weight polystyrene and polyethylene glycol.22 During the last years, the desire for highly sensitive LDIsubstrates, which are applicable for a broad range of analytes, has become more and more important, especially in the fields of peptidomics23 and metabolomics.24,25 MALDI-MS is often not the adequate analytical tool for detection of low-molecular weight compounds like amino acids, carbohydrates, small lipids, and peptides or other metabolites, although matrix suppression can be achieved under certain conditions.26 We report here the use of a special matrix-free substrate for laser desorption/ionization, based on diamond-like carbon (DLC), coated onto a molybdenum-sputtered “digital versatile disc” (12) Gorecka-Drzazga, A.; Bargiel, S.; Walczak, R.; Dziuban, J. A.; Kraj, A.; Dylag, T.; Silberring, J. Sens. Actuators, B 2004, 103, 206–212. (13) Dale, M. J.; Knochenmuss, R.; Zenobi, R. Anal. Chem. 1996, 68, 3321– 3329. (14) Ren, S.; Zhang, L.; Cheng, Z.; Guo, Y. J. Am. Soc. Mass Spectrom. 2005, 16, 333–339. (15) Hopwood, F. G.; Michalak, L.; Alderdice, D. S.; Fisher, K. J.; Willett, G. D. Rapid Commun. Mass Spectrom. 1994, 8, 881–885. (16) Kalkan, A. K.; Fonash, S. J. Mater. Res. Soc. Symp. Proc. 2003, 788, 595– 600. (17) Kim, H. J.; Lee, J. K.; Park, S. J.; Ro, H. W.; Yoo, D. Y.; Yoon, D. Y. Anal. Chem. 2000, 72, 5673–5678. (18) Kim, J.; Paek, K.; Kang, W. Bull. Korean Chem. Soc. 2002, 23, 315–319. (19) Shiea, J.; Huang, J. P.; Teng, C. F.; Jeng, J.; Wang, L. Y.; Chiang, L. Y. Anal. Chem. 2003, 75, 3587–3595. (20) Peterson, D. S.; Luo, Q.; Hilder, E. F.; Svec, F.; Frechet, J. M. J. Rapid Commun. Mass Spectrom. 2004, 18, 1504–1512. (21) Woldegiorgis, A.; von Kieseritzky, F.; Dahlstedt, E.; Hellberg, J.; Brinck, T.; Roeraade, J. Rapid Commun. Mass Spectrom. 2004, 18, 841–852. (22) Woldegiorgis, A.; Lo ¨wenhielm, P.; Bjo ¨rk, A.; Roeraade, J. Rapid Commun. Mass Spectrom. 2004, 18, 2904–2912. (23) Verhaert, P.; Uttenweiler, J. S.; de Vries, M.; Loboda, A.; Ens, W.; Standing, K. G. Proteomics 2001, 1, 118–131. (24) Seetharaman, V.; Jones, D.; David, I.; Broadhurst, J. E.; Tudor, J.; Warwick, B. D.; Hayes, A.; Burton, N.; Stephen, G. O.; Douglas, B. K.; Royston, G. Metabolomics 2005, 1, 243–250. (25) Seetharaman, V.; Gaskell, S.; Royston, G. Rapid Commun. Mass Spectrom. 2006, 20, 1192–1198. (26) Knochenmuss, R.; Dubois, F.; Dale, M. J.; Zenobi, R. Rapid Commun. Mass Spectrom. 1996, 10, 871–877.

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(DVD). DLC is a kind of amorphous carbon with mixed levels of sp3 and sp2 hybridized carbons.27 For the first time it is discovered to be an excellent material in LDI-MS, particularly for the analysis of metabolites and low-molecular weight peptides. The physical characteristics of these nanosurfaces, like thermal, electric, and chemical are contributing toward the LDI processes.28 The advantage of using DVDs as a base material lies in the additional possibility to save mass data directly onto the disk. Sample, together with data, can be stored on one device and confusions can be avoided. EXPERIMENTAL SECTION Chemicals and Reagents. All chemicals were used without further purification unless otherwise noted. Methanol (HPLC, ) 99.9%), trifluoroacetic acid (for LC-MS, ) 99.9%), fibrinopeptide B human (GluFib, ) 97%), L-sorbose () 98%), lactulose () 95%), L-phenylalanine () 98%), and deoxycholic acid () 99%) were obtained from Sigma-Aldrich. 1,2 Diheptadecanoyl-sn-glycero-3[phospho-rac-(1-glycerol)] was purchased from Avanti Polar Lipids Inc., AL. Bovine serum albumin (BSA)-digest was ordered from Bruker-Daltonics Inc. (Bremen, Germany). All water used for preparing the standard solutions was purified by a NANOpure Infinity-unit (Barnstead, Boston, MA). Digital versatile discs (1.4 GB) coated with DLC were provided by Sony DADC (Anif, Austria). Preparation of the DLC Coated DVD Matrix-Free Target. The most usual way of application for DLC is in the form of coatings on various substrates like metals and polymeric materials, depending upon the vacuum and temperature conditions. The advantage offered is that it can be coated on different substrates. The composition is based on the presence of different additives ranging from hydrogen, metals, or graphitic sp2 carbon atoms. The absence of impurities excludes the degradation of surfaces and thus offers longer shelf life. DLC coating with 35% sp3 hybridization and a thickness of approximately 300 nm was prepared by the pulsed laser deposition (PLD) technique.29 The PLD technique provides fast deposition rates and homogeneous DLC coatings. The nanogrooves are achieved through appropriate roughness, which is controlled as a function of layer thickness. The roughness was achieved either through the chemical etching or by lasers. The layer conductivity was in the range of 2 kΩ, which was measured across the surface by a simple ohmmeter. Before the DLC coating was applied, a molybdenum interlayer with a thickness of 30 nm was sputtered on top of the DVD using conventional sputtering equipment. Preparation of Standard Solutions. L-Sorbose, lactulose, and L-phenylalanine were dissolved in deionized water. TFA (0.1%) was used as a solvent in the case of BSA-digest and fibrinopeptide B. Deoxycholic acid and 1,2-diheptadecanoyl-sn-glycero-3-[phosphorac-(1-glycerol)] were dissolved in methanol. Sample Preparation. All measurements were conducted with 1 µL of each analyte solution on the DLC coated DVD. The prepared solutions were placed on the surface by pipetting out the desired solutions. No additional energy-absorbing matrix was added. (27) Robertson, J. Mater. Sci. Eng., R 2002, 37, 129. (28) Alimpiev, S.; Nikiforov, S.; Karavanskii, V.; Minton, T.; Sunner, J. J. Chem. Phys. 2001, 115, 1891–1901. (29) Bonelli, M.; Miotello, A.; Mosaner, P.; Casiraghi, C.; Ossi, P. M. J. Appl. Phys. 2003, 93, 859–865.

Figure 1. Schematic illustration of a diamond-like carbon coated DVD: (A) using the metal interlayer for laser desorption/ionization mass spectrometer and (B) the DVD (1.4 GB) can be simultaneously used as a storage device for mass spectra.

Instrumentation. All measurements were performed on a MALDI/TOF-MS (Ultraflex MALDI TOF/TOF, Bruker Daltonics, Bremen, Germany) in the reflector mode. The detector energy was set to 1623 V, and the laser frequency was adjusted to 25 Hz. Data processing was carried out by Flex analysis 2.4 post analysis software and data acquisition by Flex control 2.4. RESULTS AND DISCUSSION In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), problems arise with the routine MALDI matrixes (energy absorbing substances), which cause interfering signals, especially in the low mass range. LDI-MS is facilitated enormously with submicrometer structures, especially when working matrix-free.30 The material employed here is based on nanostructured diamond-like carbon, acting as energy absorbing material. Diamond-like-carbon is highly biocompatible, chemically inert, and therefore shows promise as a sensitive matrix-free LDI target. DLC is coated on normal digital versatile discs (1.4 GB). A thin metal interlayer of molybdenum (Mo) is present in between, to avoid the interferences from material itself, i.e., polycarbonate. The metal interlayer contributes to the advantage that it provides the appropriate conductivity required for electrical charge dissipation on the target surface. Additionally, the advantage of the metal layer lies in to protect the underneath polycarbonate substrate of the DVD target from laser effects in LDI/TOF-MS instruments, as the whole construction is acting as a MALDI target, inserted into the special adapter created for this purpose. Refocus of the laser in the machine is not required because of the exact height achieved through machining procedures. Complete demonstration of the utilized device is depicted in Figure 1a. The construction acts as a target for the incident laser in the MALDI-MS instrument shown in Figure 1b. The total number of spots range around 100, and the sample areas (a few micrometers) are surrounded by hydrophobic boundaries created by the conventional screen printing method. This adaptation provides the necessary preconcentration and helps to improve the detection limits of the described device. The purpose of DVD (storage device) in this construction is to provide a technical advantage of storing data directly on the disc. The data storing is carried out by taking the DLC coated (30) Okuno, S.; Arakawa, R.; Okamoto, K.; Matsui, Y.; Seki, S.; Kozawa, T.; Tagawa, S.; Wada, Y. Anal. Chem. 2005, 77, 5364–5369.

DVD target out of the MALDI-MS machine, followed with the processed data burning by computer software. This advantage of storage is more pronounced when the described device is employed in routine metabolomics and peptidomics where the samples can number up in the hundreds by using special robotic systems. The mechanism of laser desorption/ionization is facilitated by unique nanostructures and a large number of vacancies present in DLC films.31 These vacancies are generated by the influence of ions reaching the growing layer with enough energy, sputtering off weakly associated entities or damaging these layers. The subgap (electronic gap) absorptions, associated with the relative sp2 carbon content gives the measured absorptivity at shorter wavelengths in the range of 305-330 nm.32 The broadened solid phase absorption profiles provide the liberty to be employed for MALDI-MS analysis at laser wavelengths outside their absorption bands.33 The DLC layers possess sp2 bonded carbon atoms closer together with sp3 bonds, and the process of their mixing has a combined effect rather than to show the individual characteristics. Furthermore, sp2 and sp3 carbons (varying bond lengths) are adjusted in a way to reduce the so-called stress, which can affect proper adhesion to the substrate. That is why the Mo interlayer is deposited in between the DLC films and the substrates, to adjust the atomic spacing constraints and to reduce the stress concentrations at the coating-substrate interface. The combination of the DLC layer with the metal interlayer makes a kind of two-layer composite, which offers a sensitive platform to meet the detection limit of 10 fmol/µL for [Glu1]-fibrinopeptide B (m/z 1570.6). The detection limit for carbohydrates like L-sorbose and lactulose is even lower (around 1 fmol/µL for L-sorbose), as they are known to be the easily desorbed sugars. The sensitivity is better than that of silicon dioxide chips (50 fmol), usually termed as DIOSD (desorption/ionization on silicon dioxide) and comparable to recently developed mesoporous silicates (10 fmol), referred to as DIOM (desorption/ionization on mesoporous silicate). DLC surfaces are highly hydrophobic, resulting in the preconcentration of analyte molecules and consequently enhancing the sensitivity. Roughness plays a crucial role in the DI process, as the very smooth surfaces do not contribute toward the absorption of laser energy. Short laser pulses are used for the DI process, as longer pulses normally can disintegrate the analyte molecules in the matrix-free approach. These one-way DLC matrix-free surfaces offer high target-to-target reproducibility, and their disposability excludes any sort of memory effects in multiuse surfaces. The dependence of spectral quality and quantitative investigations on the homogeneous cocrystallization of matrix and analyte molecules (sweet spots) is also avoided, due to the absence of routine matrixes. It is also revealed that the laser energy requirement for desorption/ionization is less with the DLC-metal composite in comparison to bare DLC. The employment of low laser powers reduces the fragmentation of analytes. The mass spectra recorded with DLC produces almost no background, as compared to the spectra recorded with routine matrixes. The versatility of the DLC matrix-free target can be seen from varying classes of analytes covered by vacuum LDI-MS. The (31) Esaghi, M. A.; Shafiekhani, A. J. Phys. D: Appl. Phys. 1999, 32, L101–L104. (32) Senkevich, J. J.; Leber, D. E.; Tutor, M. J.; Heiks, N. A.; Ten, E.; Greg, A.; Scherrer, D. W., II J. Vac. Sci. Technol., B 1999, 17, 2129–2135. (33) Dreisewerd, K. Chem. Rev. 2003, 103, 395–425.

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Figure 2. Mass spectral data of two carbohydrates performed on the DLC coated DVD. Matrix-free mass spectra of 1 nmol/µL L-sorbose (m/z 180.16) (A) and 1 nmol/µL lactulose (m/z 342.30) and (B) showing their sodium and potassium adducts. The sodium (m/z 22.9) and potassium (m/z 38.9) ions were also detected. Spectra A and B were recorded in reflector mode by averaging 400 laser shots with a nitrogen laser (337 nm).

analytes constitute a group based on amino acids, carbohydrates, peptides, lipids, and metabolites. These analytes explicitly fall in the mass range from 100 to 1500 Da, where routine matrixes have a large number of interfering signals. The investigated mass range can be increased to higher limits by introducing chemical functionalities to the DLC surface. The effectivity of the matrixfree nature of the DLC surface is obvious from the signal-to-noise ratio (S/N) and isotopic resolution (R). The matrix-free mass spectra of two carbohydrates are given in Figure 2 performed on the DLC coated DVD. In the first case (A), 1 nmol/µL L-sorbose (m/z 180.16) is measured at 203.04 (Na+ adduct) and 219.05 (K+ adduct). Additionally, the presence of Na+ 7470

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Figure 3. Matrix-free mass spectra of two peptides obtained from the DLC coated DVD. (A) Analysis of 600 fmol/µL [Glu1]-fibrinopeptide B (m/z 1570.6). (B) Mass spectrum of 5 pmol/µL bradykinin fragment 1-7 (m/z 757.9). Both peptides are present in their protonated forms. The spectra shown here are the average of 400 shots with a nitrogen laser (337 nm).

(m/z 22.9) and K+ (m/z 38.9) peaks at the respective masses show the effectiveness of the matrix-free target, in terms of the lowest mass range covered (Figure 2A). In the second case (B), 1 nmol/ µL lactulose (m/z 342.30) is recorded as Na+ and K+ adducts at the masses labeled in Figure 2B. The ratio between the lactulose peaks and the Na+ and K+ peaks is rather unusual; as in LDI-MS usually a decrease in signal intensity is observed with increasing m/z ratio. Furthermore, the spectra reveal that the points on the target at which laser impingement is made are rich in these ions (Na+ and K+) in comparison to the analyte concentration (nanomoles per microliter). The broad range of analytes measured with the described DLC surfaces also covers peptides. Matrix-free mass spectra of 600 fmol/µL [Glu1]-fibrinopeptide B (m/z 1570.6) is shown in Figure

Figure 4. LDI-TOF mass spectrum of a 5 pmol/µL BSA-digest solution. Spectra were recorded for a 5 pmol/µL solution by averaging 400 laser shots using a nitrogen laser (337 nm).

3A. The mass peak at m/z 1570.886 is the mentioned protonated peptide with the resolution, R ) 10 878 and S/N ) 361.7. This is the extra feature of this DLC coated DVD matrix-free target, that there is no need to add any additive for ionization which is normally the case for many SALDI-matrixes.8 Figure 3B is based on the mass spectra of 5 pmol/µL bradykinin fragment 1-7 (m/z 757.9), observed at m/z 757.9 in the protonated form. The identification of peptides comes into the spotlight when the lowmass peptides are eluted or digested from larger proteins as a result of the biomarker MELDI identification process.34-43 The proof of this necessity is depicted from the matrix-free mass spectra of 5 pmol/µL BSA-digest solution as shown in Figure 4. The data obtained are searched against Swiss-Prot using Mascot (MatrixScience) to identify with a score greater than the 95% confidence level. Moreover metabolomics covers another range of analytes where the systematic study of unique chemical (34) Najam-ul-Haq, M.; Rainer, M.; Huck, C. W.; Stecher, G.; Feuerstein, I.; Steinmueller, D.; Bonn, G. K. Curr. Nanosci. 2006, 2, 1–7. (35) Feuerstein, I.; Rainer, M.; Bernardo, K.; Stecher, G.; Huck, C. W.; Kofler, K.; Pelzer, A.; Horninger, W.; Klocker, H.; Bartsch, G.; Bonn, G. K. J. Proteome Res. 2005, 4, 2320–2326. (36) Rainer, M.; Najam-ul-Haq, M.; Huck, C. W.; Feuerstein, I.; Bakry, R.; Huber, L. A.; Gjerde, D.; Zou, X.; Qian, H.; Du, X.; Wei-Gang, F.; Ke, Y.; Bonn, G. K. Rapid Commun. Mass Spectrom. 2006, 20, 2954–2960. (37) Feuerstein, I.; Najam-ul-Haq, M.; Rainer, M.; Trojer, L.; Bakry, R.; Aprilita, N. H.; Stecher, G.; Huck, C. W.; Bonn, G. K. J. Am. Soc. Mass Spectrom. 2006, 17, 1203–1208. (38) Najam-ul-Haq, M.; Rainer, M.; Schwarzenauer, T.; Huck, C. W.; Bonn, G. K. Anal. Chim. Acta 2006, 561, 32–39. (39) Rainer, M.; Najam-ul-Haq, M.; Bakry, R.; Huck, C. W.; Bonn, G. K. J. Proteome Res. 2007, 6, 382–386. (40) Trojer, L.; Stecher, G.; Feuerstein, I.; Bonn, G. K. Rapid Commun. Mass Spectrom. 2005, 19, 3398–3404. (41) Kloss, F. R.; Najam-ul-Haq, M.; Rainer, M.; Gassner, R.; Lepperdinger, G.; Huck, C. W.; Bonn, G.; Klauser, F.; Liu, X.; Memmel, N.; Bertel, E.; Garrido, J.; Steinmu ¨ ller-Nethl, D. J. Nanosci. Nanotechnol. 2007, 7, 1–7. (42) Najam-ul-Haq, M.; Rainer, M.; Trojer, L.; Feuerstein, I.; Vallant, R. M.; Huck, C. W.; Bakry, R.; Bonn, G. K. Exp. Rev. Proteomics 2007, 4, 447–452. (43) Vallant, R. M.; Szabo, Z.; Bachmann, S.; Bakry, R.; Najam-ul-Haq, M.; Rainer, M.; Heigl, N.; Petter, C.; Huck, C. W.; Bonn, G. K. Anal. Chem. 2007, 79, 8144–8153.

Figure 5. Analysis of metabolites on the DLC coated DVD, without using a MALDI matrix. All solutions consisted of the same concentration (1000 ppm each) with 1 µL placed on the disc. (A) Mass spectrum obtained from phenylalanine (m/z 165.2). (B) Analysis of deoxycholic acid (m/z 392.58). (C) Mass data of 1,2 diheptadecanoyl-sn-glycero3-[phospho-rac-(1-glycerol)] (m/z 773.01), demonstrating the applicability of DLC for lipids. Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Table 1. Selected m/z Values of Three Independent Measurements Showing the Reproducibility of Matrix-Free Analysis on DLC Coated DVD Targets m/z (Na+ adduct)

carbohydrates L-sorbose, m/z 180.16 lactulose, m/z 342.30

peptides [Glu1]-fibrinopeptide B, m/z 1570.6 bradykinin fragment 1-7, m/z 757.9 metabolites phenylalanine, m/z 165.2 deoxycholic acid, m/z 392.58 1,2 diheptadecanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], m/z 773.01

fingerprints from specific cellular processes is vital to measure in the human metabolome. The metabolome represents the collection of all metabolites in a biological organism, which are the end products of gene expression. Thus metabolic profiling gives an instantaneous snapshot of the cell physiology. Their small mass range is effectively measured with this matrix-free device. Figure 5 shows the matrix-free mass spectra of three metabolites measured for 1000 ppm concentration solutions. Phenylalanine (m/z 165.2, 6.0 nmol/µL), an essential amino acid which plays a role in the biosynthesis of other amino acids and related biochemical processes for the synthesis of important neurotransmitters, is measured at m/z 188.04 and 204.03 as the Na+ and K+ adducts, respectively (Figure 5A). Deoxycholic acid (m/z 392.58, 2.5 nmol/µL), used in the emulsification of fats for the absorption in the intestine of the human body, is recorded at m/z 415.29 and 431.25 again as the Na+ and K+ adducts (Figure 5B). Another class of metabolites, based on lipids, is also analyzed on the DLC coated DVD target through LDI-MS. A phospholipid, named as 1,2-diheptadecanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] at a concentration of 1.3 nmol/µL, is measured at m/z 795.56 and 811.52 as adducts (Figure 5C). The reproducibility for the device and the sample preparation is checked through quantitative treatments. The spectra recorded for every analyte on different targets at different times show similar signals but with only minor intensity differences, which can be attributed to the variation resulting from sample preparation. Furthermore, statistical tests are also applied by choosing the peaks out of recorded mass spectra. The selected mass peaks, along with their standard deviations, are given in Table 1. The data recorded produce minor standard deviations and the highest value (0.42) is found to be for [Glu1]-fibrinopeptide B (m/z 1570.6). This also supports the general trend of MALDI-MS where by increasing the mass range measured, there is slight shift in the masses with lower resolutions.

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standard deviation

203.04 365.14

203.10 365.20 m/z

203.06 365.19

0.03 0.03 standard deviation

1570.89 757.90

1570.07 757.84 m/z (Na+ adduct)

1570.67 757.96

0.42 0.06 standard deviation

188.05 415.29 795.04

188.04 415.29 795.31

188.04 415.23 795.56

0.01 0.04 0.26

CONCLUSION For the first time, it is shown that DLC based substrates are capable of absorbing laser light by transferring energy to analytes for attaining the gaseous phase, followed by their subsequent ionization. The performed experiments demonstrate that the target can be successfully applied as a sample support in LDI-MS for the analysis of a broad range of analytes like carbohydrates, peptides, amino acids, and lipids. The universal applicability, reproducibility, high performance, and sensitivity are making DLC coated LDI-supports much more competitive with the state-of-theart devices. The surface holds the advantage of higher salt tolerance, avoiding the suppression in desorption/ionization phenomenon due to salts. This claim is supported by the general observation of the dried spot, as the white colored salts are deposited on the boundary wall of the measuring area. This layerbased matrix-free system is better than suspension-based systems (carbon nanotubes, fullerenes, alumina), which generally suffer from huge mass shifts, thus requiring internal standards for mass calibration. The described matrix-free sample support provides a powerful tool for the analysis of small molecules especially in the fields of metabolomics and peptidedomics with the special advantage of simultaneous data storage on DVD substrates. ACKNOWLEDGMENT M.N.-u-H. and M.R. contributed equally to this work. This work is supported by the Austrian Science Foundation (FWF), SFBProject 021 (Vienna, Austria), the Genome Research in Austria (GEN-AU) (Federal Ministry for Education, Science and Culture, Vienna, Austria), and by the West Austrian Initiative for Nano Networking (WINN). Higher Education Commission (HEC) of Pakistan is acknowledged for providing the scholarship funding through the course of this work. Received for review June 12, 2008. Accepted July 28, 2008. AC801190E