Nanoporous Surfaces as Harvesting Agents for Mass Spectrometric

Ye Hu , Ali Bouamrani , Ennio Tasciotti , Li Li , Xuewu Liu and Mauro Ferrari ... Alessandra Luchini , Caterina Longo , Virginia Espina , Emanuel F. P...
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Nanoporous Surfaces as Harvesting Agents for Mass Spectrometric Analysis of Peptides in Human Plasma Marco Gaspari,*,†,| Mark Ming-Cheng Cheng,‡,| Rosa Terracciano,† Xuewu Liu,‡ A. Jasper Nijdam,‡ Lisa Vaccari,§ Enzo di Fabrizio,† Emanuel F. Petricoin,⊥ Lance A. Liotta,⊥ Giovanni Cuda,† Salvatore Venuta,†,# and Mauro Ferrari‡,# Department of Clinical and Experimental Medicine, Magna Græcia University, 88100 Catanzaro, Italy, Division of Hematology and Oncology, Internal Medicine, The Ohio State University, Columbus Ohio 43210, TASC-INFM CNR, S.S. 14 Km163.5, Area Science Park, I-34012 Basovizza, Trieste, Italy, and Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas Virginia 20110 Received November 23, 2005

Abstract: Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10 000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces. Keywords: peptide profiling • MALDI-TOF • nanoporous silica • nanoporous silicon • human plasma • mass spectrometry

Introduction The low-molecular weight (LMW) region of the blood proteome has gained increasing interest in recent years as a potential source of diagnostic markers for diseases.1,2 Since the introduction of the concept of mass spectrometry based protein profiling3 (MS), many dozens of research reports have appeared in the literature. Panels of peptides of potential diagnostic importance present in blood plasma/serum have been produced for diseases such as gastric cancer,4 ovarian cancer,5 colorectal cancer,6 Alzheimer’s disease.7 As research moves forward, the approach is progressively being refined with respect to various stages of the analysis. For example, focus has been recently put on sample collection/storage procedures.8,9 The methodological approach used to generate the profile of peptides and proteins generally relies on surface-enhanced * To whom correspondence should be addressed. Tel: +39 0961 3694204. Fax: +39 0961 3694090. E-mail: [email protected]. † Magna Græcia University. ‡ The Ohio State University. § TASC-INFM CNR. ⊥ George Mason University. | Sharing equally the lead authorship. # Sharing equally the senior authorship. 10.1021/pr050417+ CCC: $33.50

 2006 American Chemical Society

laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF).10 This analysis consists of performing matrixassisted laser desorption mass spectrometry (MALDI) on analytes previously immobilized on a chip surface by means of various chromatographic interaction modes (hydrophobic, ionic, metal affinity). It has been observed that in case of serum/plasma analysis, this method might suffer from the interference of highly abundant proteins, which would strongly compete with analytes present at lower concentration for the limited number of binding sites present on the surface, ultimately limiting the sensitivity of the approach to detecting species present in serum/plasma at levels in the low µg/mL range.11 The sensitivity of modern mass spectrometers strongly depends on the type of instrument and is constantly improving with time. Nevertheless, there is no doubt that MS sensitivity available today would allow detection of peptides present in plasma/serum at much lower concentration. Thus, improving selectivity of the analysis rather than MS sensitivity could possibly be a better way of looking into low-abundance species. As already addressed in the literature, an efficient way of improving selectivity could be to develop devices allowing selective isolation of LMW peptides prior to surface capture and/or MS analysis.12,13 Recently, it has been shown that the majority of the low molecular weight (e.g., < 20 kDa) information content that underpin the MS signatures exist in a complexed state, bound to highly abundant carrier proteins such as albumin.14 In fact, procedures employed in the past to selectively deplete serum and plasma of albumin were likely throwing out the vast majority of these small molecules as well. In this work, nanoporous surfaces have been used as harvesting material for LMW peptides before MALDI-TOF analysis in order to further enrich for the carrier-protein bound information archive. Human plasma was directly applied to a silica-based surface for selective peptide enrichment. After washing the surface, adsorbed molecules were extracted using classical MALDI matrix solutions. The method proved effective at isolating LMW peptides from this complex biological fluid. Preliminary results using silicon-based nanoporous surfaces applied to the analysis of a mixture of standard proteins and peptides are also shown. Journal of Proteome Research 2006, 5, 1261-1266

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Nanoporous Surfaces for MS of Plasma Peptides

Figure 1. Panel a: morphology of the nanoporous oxide layer. Panel b: schematic of the harvesting protocol consisting of (1) deposition of plasma directly onto the chip surface; (2) washing of unbound substances; (3) extraction of bound molecules; and (4) MS analysis.

Experimental Section Nanoporous Surface Fabrication. The nanoporous film of oxide was prepared as followed. A 8.71-g portion of surfactants

technical notes EO106PO70EO106 (Pluronic F127, BASF) was added in 23 g of ethanol. A mixture of 10 g of tetraethyl orthosilicate (TEOS, Aldrich), 0.1006 g of hydrochloride (20%), 10 g of Ethanol and 10.629 g of water was then added under vigorous stirring.15 After aged for 3-6 h at room temperature, the precursor solution was spin-coated onto the silicon wafer at 1900 rpm for 30 s. After spin-coating, the film was backed at 100 °C for 12 h, followed by 400 °C for 2 h in a furnace. Nanoporous silicon was fabricated by anodization of polished boron-doped silicon (resistivity 5-10 Ω‚cm) of [100] crystal orientation (supplied by SilChem Inc.), employing an electrolyte binary mixture of HF 39.5% (Carlo Erba, 152 mL) and anhydrous ethanol (JTBaker, 98 mL) at a constant density of 5.56 mA/cm2 for 7 min. Sample rinsing was performed by immersion first into deionized water, then in ethanol and pentane (Fluka). The solvent excess was removed under a nitrogen stream, and the samples were completely dried and stored in a vacuum chamber. SEM images of the samples showed and uniform porous layer 1.8 µm thick, characterized by mesopores with an average diameter of about 5 nm. Sample Preparation. Standard peptides substance P, renin substrate tetrapeptide, angiotensin I, and bovine serum albumin were from Sigma-Aldrich (St. Louis, MO). Human plasma was collected from healthy volunteers, according to published guidelines,9 under consent and Institutional Review Board monitoring for human subjects protection.

Figure 2. Harvesting of three standard peptides (concentration of 100 ng/mL each) in the presence of a 1000-fold molar excess of bovine serum albumin (5 mg/mL) using a nanoporous silica chip. MALDI-TOF analyses were performed in CHCA matrix (left panels) and SA matrix (right panels). Panels a,b: supernatant after exposure to chip surface (sample/matrix ratio 1:9). Panels c,d: analysis of first three washings (pooled), sample/matrix ratio 1:9. Panels e,f: analysis of the last two washings (pooled), sample/matrix ratio 1:3. Panels g,h: analysis of chip extracts, sample/matrix ratio 1:3. 1262

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Figure 3. MALDI-TOF profiles of human plasma using as matrixes CHCA (left panels) and SA (right panels). Panels a,b: control experiment without chip incubation (direct MS analysis with no pretreatment); a plasma aliquot was diluted 100-fold with matrix for MS analysis analysis. Panels c,d: control experiment using a solid silica surface (nonporous). Panels e,f: analysis of human plasma proteins after exposure to a nanoporous silica chip. All experiments used 5 µL of human plasma spiked with calcitonin at a concentration of 1 µg/mL. For obtaining spectra c and e, chip surfaces were extracted directly with matrix solution; for spectra d and f, the extract was instead mixed with matrix in a subsequent step (matrix/extract ratio 3:1). The calcitonin peak, only visible in panel “e”, is marked with a star.

The chip surfaces were wetted using 2-propanol (HPLCgrade, Merck, Darmstad, Germany). After a water wash, (HPLCgrade, Merck), 5 µL of sample solution were applied to the chip surface and allowed to incubate for 30 min at room temperature in 100% humidity. The sample was removed by using a pipettor. The surface was then washed by 5 sequential 5 µL aliquots of water, allowing the droplet to rest on the surface for 1 min each time. Supernatant and washings were stored and subsequently mixed with the appropriate amount of matrix for MS analysis. After the last washing, bound species were either (i) eluted with 5 µL of extraction solution, a 1:1 mixture of acetonitrile (HPLC-grade, Merck) and 0.1% trifluoroacetic acid (TFA, ACS grade, Sigma) (v/v), and then mixed with the appropriate matrix (unless specified, matrix/sample volume ratio was 3:1), or (ii) directly extracted in matrix solution, 3 mg/ mL R-cyano-4-hydroxycinnamic acid (CHCA, Sigma) in a 1:1 mixture of acetonitrile and 0.1% TFA (v/v). For the analysis of high molecular weight proteins, a saturated solution of trans3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) in a 2:1 mixture of acetonitrile and 0.1% TFA (v/v) was used. 1 µL of the matrix/sample mixture was deposited on a MALDI sample plate and allowed to dry before mass spectrometric analysis. Mass Spectrometry. MALDI-TOF was performed on a Voyager DE-STR MALDI-TOF (Applied Biosystems, Framingham, MA) mass spectrometer equipped with a nitrogen laser emitting at 337 nm. Spectra were acquired in linear positive mode using a delayed extraction time of 700 nanoseconds and an accelerating voltage of 20 kV. 500-600 laser shots were typically averaged to produce the final sample spectrum. Spectra processing was performed by using the Data Explorer software (Applied Biosystems).

Results and Discussion The work herein described aimed at developing an approach for isolating LMW peptides based on the size-exclusion principle. To achieve this goal, a nanoporous silicon chip surface, having the right porosity (few nanometers) to operate a molecular cutoff, was used. The device was fabricated by coating silicon chips with a 500 nm thick nanoporous film of silicon oxide. On the basis of Lorentz-Lorenz model, the refractive index was measured in order to determine the porosity of the film using ellipsometry. It was estimated that a porosity of 57% was created. Brunauer-Emmett-Teller (BET) surface area was 670m2/g using nitrogen adsorption-adsorption isotherms measurements. Average pore size was estimated to be about 7 nm. Panel “a” of Figure 1 shows the morphology of the film using Transmission Electron Microscopy (TEM, Philips CM200) operating at acceleration voltage 200 kV with magnification 310 K. The sample was lifted-off from the wafer using Focus Ion Beam (FIB, FEI DB235), and placed on a standard copper grid. The fabricated chips were subsequently used for LMW peptide harvesting. A schematic of the protocol used, as described in the Experimental Section, is presented in Figure 1 panel “b”. Briefly, after wetting the surface with sequential washes of 2-propanol and deionized water, a droplet of the analyte solution was pipetted directly onto the chip surface and let it incubate for capturing protein/peptide species. Following a series of sequential washings in deionized water, bound species were released from the surface by the addition of an acidic solution containing a high percentage of organic solvent (50% v/v). To assess the efficacy of size selection, an experiment using three small standard peptides and bovine serum albumin was performed. An aqueous solution of the three peptides (100 ng/mL each, buffered at pH 7.4 with 5 mM tris) in the presence Journal of Proteome Research • Vol. 5, No. 5, 2006 1263

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Table 1. Variation Coefficients for Ten Selected Peaks Belonging to the Spectra Shown in Figure 4a m/z

average normalized rel intensity (%)

CV

1062.8 1533.6 906.5 3437.9 2368.9 1742.9 4579.8 2512.8 7773.6 2431.1

100 9.8 6.5 3.0 1.6 1.4 0.8 0.5 0.4 0.4

3.5 5.7 11.5 12.7 6.8 14.3 19.1 10.3 13.4 8.3

a Ten MALDI-TOF analyses obtained from 5 replicate preparations of the same plasma sample were considered.

Figure 4. Repeatability of MALDI-TOF peptide profiles obtained by using nanoporous silicon oxide chip harvesting.

of a 1000-fold molar excess of bovine serum albumin (Mw ∼66 kDa, 5 mg/mL) was applied onto the chip surface and subjected to the extraction protocol. Figure 2 shows MALDI-TOF analyses of supernatant, washings and extracts relative to this experiment. Both CHCA and SA matrixes were used in order to operate in optimal conditions for the whole Mw range of MS analysis. The experiment clearly demonstrated the selective capture of the three LMW peptides and their subsequent release in the extract. The presence of the high Mw albumin was exclusively detected in the supernatant after exposure to the chip surface and in the first washings, while there was no trace of it in the last washing solutions and extraction solutions (panels “f” and “h”). Matrix/sample ratios were higher for the supernatant and first washings in order to achieve optimal matrix crystallization, otherwise disturbed by the excessive protein content of the preparation. Experiments on blood plasma peptide harvesting were also carried out. After exposure to the silica nanoporous surface and 5 replicate washings, only LMW peptides were present in the recovered extracts, as shown in Figure 3, panels “e” and “f”, which display the MALDI-TOF spectra of the extracts covering both low- and high-molecular weight range. Control experiments using a nonporous silica surface yielded no peptides or proteins in the final extract, as documented in panels “c” and “d”. Direct analysis of plasma proteins or peptides without any pretreatment gave very abundant signals for the high m/z 1264

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Figure 5. LMW harvesting of plasma spiked with human calcitonin. Four experiments on plasma spiked with decreasing calcitonin concentration are shown (zoom on the m/z window around the calcitonin peak). (i) 1000 ng/mL, (ii) 200 ng/mL, (iii) 50 ng/mL, and (iv) 20 ng/mL. Incubation and MS conditions were as described in the experimental. Direct extraction in matrix solution was performed.

region of the spectrum, as predicted, with the signal from albumin largely predominant. On the other hand, only a few weak signals were observed from the LMW end of the spectrum, though the sample had to be diluted 100-fold in matrix to achieve acceptable matrix crystallization. In Figure 3, panel “e”, about 70 LMW peptides were detected, including human calcitonin, which had been spiked in the incubated plasma at

technical notes

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Figure 6. Harvesting of three standard peptides (concentration of 100 ng/mL each) in the presence of a 1000-fold molar excess of bovine serum albumin (5 mg/mL) using a nanoporous silicon chip. MALDI-TOF analyses were performed in CHCA matrix (left panels) and SA matrix (right panels). Panels a,b: supernatant after exposure to chip surface (sample/matrix ratio 1:9). Panels c,d: analysis of first three washings (pooled), sample/matrix ratio 1:9. Panels e,f: analysis of the last two washings (pooled), sample/matrix ratio 1:3. Panels g,h: analysis of chip extracts, sample/matrix ratio 1:3.

a concentration of 1 µg/mL (calcitonin had been also spiked in both control samples, and no signal could be detected in the corresponding MALDI-TOF spectra). The cutoff mass value achieved by the actual experimental conditions and nanoporosity of the surface employed was estimated to be 15 kDa. Isolation and detection of LMW peptides as displayed in Figures 2 and 3 is to ascribe to the specific nanometer-sized porosity of the chip surface. Only analytes small enough to easily penetrate the pores can be quantitatively adsorbed onto the chip surface. Analyte adsorption on the chip surface has to be strong enough to resist extensive washes of the chip after capture during the incubation step. In this particular case, ionic interactions of the LMW analytes with the silanol groups present on the silica nanoporous layer account for analyte binding. To assess repeatability of the profile generated, five replicate analyses on the same plasma sample were performed. MALDITOF spectra of the extracts from the 5 replicates are shown in Figure 4 (raw data are shown), where the repeatability of the LMW profile can be appreciated. A duplicate MALDI-TOF acquisition of the 5 preparations produced a data set of 10 spectra which was subsequently used for statistical analysis. After baseline correction, spectra were processed using the default smoothing of the vendor’s software, which used a selfadjusting Gaussian filter, based, in this case, on the expected resolution of 2000. Subsequently, peaks were annotated in automated fashion and their m/z and intensity recorded. For

each sample, peak intensities were normalized to the total ion current of all detected peaks and expressed as % of the base peak. Peak lists of the 10 separate analyses were manually matched in order to obtain a final list of 50 peaks annotated in all 10 samples. Peak finding criteria were quite stringent, so some small intensity peaks were missed by the automated peak extraction routine. On the 50 peaks considered, an average CV of 11.8% was found for the set of 10 (5 × 2) replicates. CV values for 10 selected peaks, spanning more than 2 orders of magnitude in intensity, can be found in Table 1. Peptide profiling by mass spectrometry has been questioned for its ability to detecting low-concentration analytes in plasma/ serum.11 Unfortunately, these commentaries failed to take into consideration the ability of in vivo amplification via carrier protein binding14 as well as the ability to engineer a specific approach that can selectively sequester the low molecular weight analytes. We now understand that MS based profiling of this MW range can indeed detect very low analyte concentrations bases on this enrichment and sequestration. To estimate the detection limit (DL) of the approach here described, a peptide, human calcitonin, was spiked at different concentration levels into human plasma before analysis in order to mimic conditions in vivo. The analysis was performed by incubating the spiked plasma on a nanoporous silica chip followed by subsequent MALDI-TOF profiling. Figure 5 displays the intensity of the peak corresponding to the calcitonin Journal of Proteome Research • Vol. 5, No. 5, 2006 1265

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protonated molecule (theoretical m/z ) 3421.0) at four different concentration levels, down to 20 ng/mL. This concentration DL, though still rather high, represents a dramatic improvement with respect to recent data reported in the literature12 using similar approaches. The effect of endogenous carrier protein based amplification was not considered here since an exogenously administered analyte was analyzed. Recent reports of sequenced carrierprotein bound LMW information for ovarian cancer detection revealed analytes of ultralow abundance, or which had never been known to exist in the circulation before.16,17 It is interesting to notice that the drop of spiked plasma having the lowest calcitonin concentration, 20 ng/mL, contained an absolute amount of 100 pg calcitonin (5 µL applied to the nanoporous surface). Considering that 1/3 of the extraction solution was deposited on the MALDI plate for analysis, a maximum of 33 pg calcitonin was available for MALDI-TOF detection. Such amount corresponds to the actual LD for calcitonin when analyzed by MALDI-TOF in standard conditions (i.e., a 1 µL mixture of a peptide solution plus CHCA matrix deposited on a MALDI target for analysis). This indicates that a significant portion of the spiked calcitonin was efficiently harvested from plasma by the nanoporous surface even at the lowest concentration analyzed, and made available for MS detection after LMW peptide extraction. The experiments showed in Figure 2 (using a solution of standard peptides) and Figure 5 indicate the ability of the described protocol in achieving ng/mL limits for the detection of peptides in solution. For biological samples, such detection levels are also reachable, as here demonstrated for calcitonin, as long as the affinity of the analyte for the chip surface is superior to its affinity for the carrier proteins present in the biological matrix. Preliminary experiments on standard mixtures have shown that also silicon-based nanoporous surfaces can achieve selective capture of peptides based on size selection. Nanoporous silicon was fabricated as described in the experimental section, creating a 1.8-µm thick mesoporous layer characterized by an average pore diameter of about 5 nm.18 The material was used to perform the same experiment on pepide standards as described above for the silica-based material. Results reported in Figure 6 demonstrate comparable efficacy of nanoporous silicon in creating a molecular cutoff and selectively extracting the three small peptides in a 1000-fold molar excess of albumin. Experiments on biofluid matrixes such as human plasma are underway. The possibility of relying on different materials promises high flexibility in terms of designing nanoporous surfaces having different porosities and adsorbing characteristics, besides the option of exploring matrix-free laser desorption as an alternative for sample ionization, as it will be discussed in the following section.

Conclusions A method for efficiently and selectively capturing lowmolecular weight peptides present in human plasma using nanoporous silicon has been demonstrated. Captured peptides present in plasma at concentration levels in the ng/mL range were profiled by MALDI-TOF mass spectrometry. Further improvements of the method aiming at increasing its sensitivity will be directed at miniaturization and automation of the sample preparation procedure. Furthermore, improvements in sensitivity could be obtained by additional sample preparation

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steps, such as performing a priori carrier protein binding as an affinity step sample preparation, followed by nanoporous surface capture and MS analysis on the same chip. Indeed, very high sensitivity mass spectrometric analyses have been recently obtained by performing laser desorption directly from derivatized porous silicon.19,20 Such an approach could allow for the rapid ability to profile, sequence and characterize the low molecular weight region of the circulatory proteome for disease-related biomarkers. In the future, these tiny biomarkers, most likely specific fragments of larger intact molecules produced by specific biological processes, can be measured by immuno-MS, MS alone, or panels of traditional immunologic detection methods. Such multiplexing holds the promise of increased clinical sensitivity and specificity over single biomarkers alone.

Acknowledgment. M.M.C.C., A.J.N., and M.F. would thank Federal Funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO12400. This work has been supported by grants from MIUR (Cofin 2003), Ministero della Salute and AIRC. References (1) Liotta, L. A.; Ferrari, M.; Petricoin, E. F. Nature 2003, 425, 905. (2) Villanueva, J.; Tempst, P. Nature 2004, 430, 611. (3) Petricoin, E. F.; Ardekani, A. M.; Hitt, B. A.; Levine, P. J.; Fusaro, V. A.; Steinberg, S. M.; Mills, G. B.; Simone, C.; Fishman, D. A.; Kohn, E. C.; Liotta, L. A. Lancet 2002, 359, 572-577. (4) Ebert, M. P.; Meuer, J.; Wiemer, J. C.; Schulz, H. U.; Reymond, M. A.; Traugott, U.; Malfertheiner, P.; Rocken, C. J. Proteome Res. 2004, 3, 1261-1266. (5) Zhang, Z.; Bast, R. C., Jr; Yu, Y.; Li, J.; Sokoll, L. J.; Rai, A. J.; Rosenzweig, J. M.; Cameron, B.; Wang, Y. Y.; Meng, X. Y.; Berchuck, A.; Van Haaften-Day, C.; Hacker, N. F.; de Bruijn, H. W.; van der Zee, A. G.; Jacobs, I. J.; Fung, E. T.; Chan, D. W. Cancer Res. 2004, 64, 5882-5890. (6) Chen, Y. D.; Zheng, S.; Yu, J. K.; Hu, X. Clin. Cancer Res. 2004, 10, 8380-8385. (7) Carrette, O.; Demalte, I.; Scherl, A.; Yalkinoglu, O.; Corthals, G.; Burkhard, P.; Hochstrasser, D. F.; Sanchez, J. C. Proteomics 2003, 3, 1486-1494. (8) Villanueva, J.; Philip, J.; Chaparro, C. A.; Li, Y.; Toledo-Crow, R.; DeNoyer, L.; Fleisher, M.; Robbins, R.; Tempst, P. J. Proteome Res. 2005, 4, 1060-1062. (9) Hulmes, J. D.; Bethea, D.; Ho, K.; Huang, S. P.; Ricci, D. L.; Opiteck, G. J.; Hefta, S. A. Clin. Proteomics 2004, 1, 17-32. (10) Issaq, H. J.; Conrads, T. P.; Prieto, D. A.; Tirumalai, R.; Veenstra, T. D. Anal. Chem. 2003, 75, 148A-155A. (11) Diamandis, E. P. Mol. Cell. Proteomics 2004, 3, 367-378. (12) Diamandis, E. P.; van der Merwe, D. E. Clin. Cancer Res. 2005, 11, 963-965. (13) Geho, D. H.; Lahar, N.; Ferrari, M.; Petricoin, E. F.; Liotta, L. A. Biomed. Microdevices 2004, 6, 231-239. (14) Mehta, A. I.; Ross, S.; Lowenthal, M. S.; Fusaro, V.; Fishman, D. A.; Petricoin, E. F.; Liotta, L. A. Dis. Markers 2003-2004, 19, 1-10. (15) Cohen, M. H.; Melink, K.; Boiarski, A. A.; Ferrari, M.; Martin, F. J. Biomed. Microdevices 2003, 5, 253-259. (16) Liotta, L. A.; Lowenthal, M.; Mehta, A.; Conrads, T. P.; Veenstra, T. D.; Fishman, D. A.; Petricoin, E. F. J. Natl. Cancer Inst. 2005, 97, 310-314. (17) Lowenthal, M. S.; Mehta, A. I.; Frogale, K.; Bandle, R. W.; Araujo, R. P.; Hood, B. L.; Veenstra, T. D.; Conrads, T. P.; Goldsmith, P.; Fishman, D.; Petricoin E. F.; Liotta, L. A. Clin. Chem. 2005, 51, 1933-1945. (18) Vaccari, L.; Canton, D.; Zaffaroni, N.; Villa, R.; Tormen, M.; di Fabrizio, E. Microelectron. Eng., in press. (19) Trauger, S. A.; Go, E. P.; Shen, Z.; Apon, J. V.; Compton, B. J.; Bouvier, E. S.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2004, 76, 4484-4489. (20) Go, E. P.; Apon, J. V.; Luo, G.; Saghatelian, A.; Daniels, R. H.; Sahi, V.; Dubrow, R.; Cravatt, B. F.; Vertes, A.; Siuzdak, G. Anal. Chem. 2005, 77, 1641-1646.

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