Monolithic Porous Polymer Layer for the Separation of Peptides and

Plates for thin-layer chromatography (TLC) with an at- tached layer of porous polymer monolith have been prepared and used for the separation of small...
0 downloads 0 Views 673KB Size
Download PDF
Anal. Chem. 2007, 79, 486-493

Monolithic Porous Polymer Layer for the Separation of Peptides and Proteins Using Thin-Layer Chromatography Coupled with MALDI-TOF-MS Rania Bakry,*,† Gu 1 nther K. Bonn,† Dieudonne Mair,‡ and Frantisek Svec‡

Institute of Analytical Chemistry and Radiochemistry, Leopold Franzens University, 6020 Innsbruck, Austria, and Molecular Foundry, E.O. Lawrence Berkeley National Laboratory, Berkeley, California 94720-8139

Plates for thin-layer chromatography (TLC) with an attached layer of porous polymer monolith have been prepared and used for the separation of small molecules, peptides, and proteins. The 50-200-µm. thin poly(butyl methacrylate-co-ethylene dimethacrylate) layers were prepared in situ using UV-initiated polymerization. Precise control of the reaction conditions enables the preparation of monolithic layers with a well-defined porous structure that determines the chromatographic performance. Compared to conventional TLC and high-performance TLC using precoated layers based on silica, the small layer thickness and absence of any binder is expected to improve both retention characteristics and separation efficiency of the polymer-based monolithic thin-layer chromatographic plates. Spots of the separated compounds were first detected using typical UV imaging. Since the monolithic thin layers can be also prepared directly on the stainless steel MALDI carrier plate, the separation in TLC format can be coupled with MALDI-TOF-MS. Application of a conventional MALDI matrix facilitated desorption and ionization of peptides and proteins for molecular weight determination of the separated compounds. At the age of almost 70 years, thin-layer chromatography (TLC) is one of the oldest chromatographic modes.1 Due to its simplicity, TLC is still widely used in a variety of fields including organic pharmaceutical laboratories to follow progress of syntheses, assess product purity, and select the mobile phase suitable for the flash chromatographic separation of the desired product. It is also used for fast and simple quality inspection, separation of rare earth metals, in athlete drug testing, clinical diagnostics, forensics tests, pesticide residue analyses, and many other fields. In the original implementation, sample is spotted near one end of the plate that is then placed in a shallow reservoir containing the mobile phase. Driven by capillary forces, the mobile phase advances vertically and the sample components are separated according to a dif* To whom correspondence should be addressed. Phone: +435125075125. Fax: +435125072965. E-mail: [email protected]. † University of Innsbruck. ‡ Molecular Foundry. (1) Poole, C. F. The Essence of Chromatography; Elsevier: Amsterdam, 2003.

486 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

ferential partitioning between the mobile phase and solid phase of the thin layer. Since the layers are most often manufactured from bare silica, the prevailing TLC mode is normal phase although bonded reversed-phase silica plates are also commercially available. Once separated, the individual spots must be visualized either directly via a chemical reaction producing compounds detectable in UV or visible light or indirectly by observing spots where fluorescence of the plate is shadowed. Although this simple approach is vital, information related only to the retardation factor (Rf value) is not sufficient to assign any structure to compound residing in the spot. Therefore, the sample has to be reanalyzed typically using an HPLC-MS technique. This procedure may require a tedious method development. In order to obtain the desired molar mass information without reanalyzing the sample, significant efforts have been invested in the development of mass spectrometric techniques enabling online analyses in the TLC-MS mode.2 Since the two-dimensional shape is characteristic of both TLC and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry plates, MALDI MS became an interesting option for direct coupling with TLC.3-5 Using this approach, a variety of compounds such as drugs,6-11 small organic molecules,12-15 lipids,16,17 oligosaccharides,18 nucle(2) Busch, K. L. J. Chromatogr., A 1995, 692, 275-90. (3) Gusev, A. I.; Proctor, A.; Rabinovich, Y. I.; Hercules, D. M. Anal. Chem. 1995, 67, 1805-14. (4) Gusev, A. I. Fresenius J. Anal. Chem. 2000, 366, 691-700. (5) Crecelius, A.; Clench, M. R.; Richards, D. S. PharmaGenomics 2004, 4, 36-8, 40, 42. (6) Crecelius, A.; Clench, M. R.; Richards, D. S.; Nichols, G.; Parr, V. Adv. Mass Spectrom. 2001, 15, 695-6. (7) Crecelius, A.; Clench, M. R.; Richards, D. S.; Parr, V. J. Pharm. Biomed. Anal. 2004, 35, 31-9. (8) Crecelius, A.; Clench, M. R.; Richards, D. S.; Parr, V. J. Chromatogr., A 2002, 958, 249-60. (9) Crecelius, A.; Clench, M. R.; Richards, D. S.; Mather, J.; Parr, V. J. Planar Chromatogr. 2000, 13, 76-81. (10) Santos, L. S.; Haddad, R.; Hoehr, N. F.; Pilli, R. A.; Eberlin, M. N. Anal. Chem. 2004, 76, 2144-7. (11) Nicola, A. J.; Gusev, A. I.; Hercules, D. M. Appl. Spectrom. 1996, 50, 147982. (12) Mehl, J. T.; Gusev, A. I.; Hercules, D. M. Chromatographia 1997, 46, 35864. (13) Mehl, J. T.; Hercules, D. M. Anal. Chem. 2000, 72, 68-73. (14) Chen, Y. C. Rapid Commun. Mass Spectrom. 1999, 13, 821-5. (15) Vermillion-Salsbury, R. L.; Hoops, A. A.; Gusev, A. I.; Hercules, D. M. Int. J. Environ. Anal. Chem. 1999, 73, 179-90. 10.1021/ac061527i CCC: $37.00

© 2007 American Chemical Society Published on Web 11/22/2006

Figure 1. SEM micrographs of porous structure of 150-µm-thick poly(butyl acrylate-co-ethylene dimethacrylate) monolith attached to a glass plate.

otides,19 peptides, and proteins3,20 have already been separated and detected. Gusev et al. were the first to demonstrate separations of proteins and peptides in a TLC format with MALDI detection. However, they found that the TLC-MALDI detection limit was several orders lower than conventional MALDI and even lower than “classical” UV detection.3 They found that adequate sensitivity was achieved only after concentrating the analyte on the surface via methanol extraction followed by addition of a conventional MALDI matrix. Yet, the separation of even small proteins remained poor probably due to unsuitable properties of the sorbent layer.3

The first monolithic, silica-based, ultra-thin-layer (UTLC) plates for the separation in the typical TLC format were introduced by Merck (Darmstadt, Germany) in 2002.21 These 6 × 3.6 cm plates carry a monolithic layer with a thickness of 10 µm. This configuration shortens the analysis times and decreases consumption of the mobile phase used for the development. However, highefficiency separations were demonstrated only with small molecules.22 Recently, these thin layers were also used in combination with MALDI detection.23 In contrast to silica-based monoliths, the rigid porous polymer monoliths are best suited for the separation of large molecules such as nucleic acids, peptides, and proteins.24 Interestingly, these

(16) Ivleva, V. B.; Sapp, L. M.; O’Connor, P. B.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2005, 16, 1552-60. (17) Ivleva, V. B.; Elkin, Y. N.; Budnik, B. A.; Moyer, S. C.; O’Connor, P. B.; Costello, C. E. Anal. Chem. 2004, 76, 6484-91. (18) Dreisewerd, K.; Koelbl, S.; Peter-Katalinic, J.; Berkenkamp, S.; Pohlentz, G. J. Am. Soc. Mass Spectrom. 2006, 17, 139-50.

(19) Isbell, D. T.; Gusev, A. I.; Taranenko, N. I.; Chen, C. H.; Hercules, D. M. Fresenius’ J. Anal. Chem. 1999, 365, 625-30. (20) Gusev, A. I.; Vasseur, O. J.; Proctor, A.; Sharkey, A. G.; Hercules, D. M. Anal. Chem. 1995, 67, 4565-70. (21) Hauck, H. E.; Schulz, M. J. Chromatogr. Sci. 2002, 40, 550-2. (22) Hauck, H. E.; Schulz, M. Chromatographia 2003, 57, S/313-S/315.

Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

487

Figure 2. TLC separation of methylene blue and methyl red on 50-µm-thick poly(butyl acrylate-co-ethylene dimethacrylate) monolithic layer attached to a glass plate using ethyl acetate-ethanol-water mixture (6:4:3) as the mobile phase (A) and MALDI spectrum from the spot of methylene blue obtained “from MALDI plate” without using any matrix (B).

stationary phases, developed in the late 1980s, were first prepared by thermally initiated polymerization in a flat mold as thin plates.25,26 Disks were then punched from these plates and used for the separations in the cross-flow mode. A similar approach was used recently to prepare thin layers of porous polymer monolith using photoinitiated polymerization.27 Despite the overwhelming popularity of TLC in the separation of small molecules, there are only a few reports concerning separation of peptides and proteins.3,20,28-32 Since two-dimensional separations are easy to achieve using TLC,33,34 it has the potential to compete with 2-D gel electrophoresis, a method currently mostly applied in proteomic studies. While the latter operates with entirely aqueous electrolytes, any combination of solvents can be used for the development in TLC. Therefore, even membrane proteins and large biopolymers are likely to be separated using this method. The objective of this report is to demonstrate for the first time the preparation of thin porous polymer monolith layers attached to a glass slide or MALDI target plate and their (23) Salo, P. K.; Salomies, H.; Harju, K.; Ketola, R. A.; Kotiaho, T.; Yli-Kauhaluoma, J. I.; Kostiainen, R. J. Am. Soc. Mass Spectrom. 2005, 16, 906-15. (24) Svec, F.; Huber, C. G. Anal. Chem. 2006, 78, 2100-7. (25) Tennikova, T. B.; Svec, F.; Belenkii, B. G. J. Liq. Chromatogr. 1990, 13, 63-70. (26) Tennikova, T. B.; Bleha, M.; Svec, F.; Almazova, T. V.; Belenkii, B. G. J. Chromatogr. 1991, 555, 97-107. (27) Rohr, T.; Hilder, E. F.; Donovan, J. J.; Svec, F.; Fre´chet, J. M. J. Macromolecules 2003, 36, 1677-84. (28) Morris, C. J. O. R. J. Chromatogr. 1964, 16, 167-75. (29) Bauer, K. J. Chromatogr. 1968, 32, 529-42. (30) Bauer, K. J. Chromatogr. 1968, 35, 538-48. (31) Bhushan, R.; Mahesh, V. K.; Mallikharjun, P. V. Biomed. Chromatogr. 1989, 3, 95-104. (32) Luo, Q.; Andrade, J. D.; Caldwell, K. D. J. Chromatogr., A 1998, 816, 97105.

488 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

use for TLC separation of proteins and peptides with on-layer detection by MALDI-TOF MS. EXPERIMENTAL SECTION Materials. Commercial grades of butyl methacrylate, ethylene dimethacrylate, styrene, divinylbenzene (65%), 2,2-azobisisobutyronitrile, 2,2′-dimethoxy-2-phenylacetophenone, 1-decanol, cyclohexanol, tetrahydrofuran, [Sar1,Ile8]-angiotensin II, angiotensin II human acetate, angiotensin I human acetate hydrate, insulin (bovine pancreas), cytochrome c (horse heart), lysozyme (chicken egg white), and myoglobin (horse heart) were all purchased from Sigma-Aldrich (Steinheim, Germany). Acrylate monomers were passed through a layer of basic alumina to remove the inhibitors, followed by distillation under reduced pressure. Styrene and divinylbenzene were distilled under reduced pressure before use. R-Cyano-4-hydroxycinnamic acid (HCCA), sinapinic acid, methylene blue, methyl red, and fluorescamine were from Fluka (Buchs, Switzerland). Acetonitrile (ACN) and trifluoroacetic acid (TFA, analytical reagent grade) were obtained from Merck. Caution: Several methacrylate- and styrene-based monomers, as well as numerous solvents and 2,2-dimethoxy-2-phenylacetophenone are known sensitizing agents. Proper precautions should be taken to avoid contact during the physical handling of these materials. Surface Modification of Glass Plates. The microscope glass plates (76 × 26 mm, 1 mm thick, MEnzel-Glaeser) were rinsed with acetone and water, activated with 0.2 mol/L sodium hydrox(33) Gocan, S. J. Liq. Chromatogr. 2004, 27, 1105-13. (34) Kalasz, H.; Hunyadi, A.; Bathori, M. J. Liq. Chromatogr. 2005, 28, 248997.

Figure 3. TLC separation of a mixture of peptides labeled with fluorescamine on 50-µm-thick poly(butyl acrylate-co-ethylene dimethacrylate) monolithic layer attached to a glass plate using 0.1 vol % TFA in 45 vol % aqueous acetonitrile as the mobile phase (A). Sample volume 0.5 µL. MALDI-TOF MS spectra of fluorescently labeled [Sar1,Ile8]-angiotensin II (B), angiotensin II (C), and neurotensin (D) obtained “from-plate” using HCCA as matrix.

ide for 30 min, washed with water followed by 0.2 mol/L HCl for 30 min, then with water again, and finally with acetone. The plates were dried at 60 °C for 1 h. Their surface activation was achieved by immersion in a 20 vol % solution of 3-(trimethoxysilyl)propyl methacrylate in 95% ethanol adjusted to pH 5 using acetic acid. The activation proceeded with occasional shaking at room temperature for 2 h. The modified plates were washed with ethanol and dried in vacuum at room temperature for 24 h.

Preparation of the Monolithic Layer. The polymerization mixture used in this study consisted of butyl methacrylate (24 wt %), ethylene dimethacrylate (16 wt %), 1-decanol (40 wt %), cyclohexanol (20 wt %), and 2,2-dimethoxy-2-phenylacetophenone (1 wt % with respect to monomers). This mixture was first deaerated by purging with nitrogen for 5 min. A Teflon gasket with a desired thickness ranging from 50 to 200 µm was placed on the top of the modified glass plate or the stainless steel MALDI Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

489

Figure 4. TLC separation of mixture of proteins labeled with fluorescamine (A). Sample volume 0.5 µL. Peaks: insulin (1), cytochrome c (2), lysozyme (3), and myoglobin (4). MALDI-TOF MS spectra of fluorescently labeled insulin (B), and myoglobin (C) obtained “from MALDI plate” using sinapinic acid as matrix.

Table 1. Repeatability of the Protein Separation Using Monolithic Thin Layers

a

protein

Rf a

SD

RSD, %

insulin cytochrome c lysozyme myoglobin

0.836 0.362 0.286 0.205

0.023 0.023 0.006 0.007

2.753 6.299 2.024 3.554

Mean value calculated from data measured for 10 spots on 5 plates.

target plate, covered with another glass plate, and clamped. The assembled mold was filled with the deaerated polymerization mixture using a syringe and exposed to UV light for 15 min using a 8-W, 254-nm lamp (CAMAG, Muttenz, Switzerland). Once the polymerization process was complete, the mold was disassembled. The monoliths attached to the plate surface were first washed with a stream of methanol. After this initial rinsing, the plates were placed in a beaker, covered with methanol, and left overnight to allow for complete extraction of the porogens. Finally, the plates were dried in vacuum at room temperature for several hours. 490 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

Sample and Matrix Solutions. Stock solutions of standard peptides and proteins (0.5 mg/mL) were prepared in 0.1 vol % TFA solution in 1:1 acetonitrile-water. These solutions were further diluted for the MALDI measurements. Methylene blue and methyl red solutions with a concentration of 0.3 mg/mL were prepared by dissolution in 80% ethanol. Saturated MALDI matrix solutions were prepared in 1:1 acetonitrile-water containing 0.1 vol % TFA. Fluorescamine (3 mg/mL) was dissolved in acetone and this solution kept at 4 °C. Fluorescent labeling of proteins and peptides was achieved after mixing their solution with an equal volume of fluorescamine solution. Labeled compounds were kept in the dark in an ice bath before loading on the TLC plates. No decrease in fluorescence was observed even after several hours. TLC Separation of Small Molecules. Mixture of methylene blue and methyl red solutions (0.2 µL) was spotted onto the monolithic TLC plate. Ethyl acetate-ethanol-water (3:2:1) was used as the mobile phase. The developing tank was conditioned for 30 min before the plate was developed. After development, the plates were air-dried and scanned with MALDI. TLC Separation of Proteins and Peptides. A sample (0.5-1 µL) was spotted onto the monolithic TLC plate and 0.1 vol % TFA

Figure 5. MALDI spectra of unlabeled cytochrome c (A), lysozyme (B), and myoglobin (C) separated on 50-µm-thick poly(butyl acrylate-coethylene dimethacrylate) monolithic layer attached to a glass plate using 0.1 vol % TFA in 60 vol % aqueous acetonitrile as the mobile phase obtained “from-plate” using sinapinic acid as matrix.

solutions in 40 or 55% aqueous ACN were used as mobile phase in the case of peptides and proteins, respectively. The developing tank was again conditioned for 30 min prior to plate development. After development, the plates were air-dried and the fluorescently labeled separated peptides were visualized by UV illumination at 366 nm. Two different protocols were used for the application of the 20 mg/mL solution of cyano-4-hydroxycinnamic acid or sinapinic acid (MALDI matrixes) in 0.1 vol % TFA in 50% aqueous ACN. In the “spotting” protocol, matrix solution (0.2-1.0 µL) were applied next to the marked spots. The matrix was rapidly adsorbed by the porous monolith and diffused, producing wetted 1-2-mmdiameter spots that were allowed to dry at room temperature. In the “spray” protocol, matrix solution was uniformly sprayed over the entire TLC plate until sufficient wetting of the plate was achieved. Instrumentation. MALDI-TOF measurements were carried out by Ultraflex I MALDI-TOF/TOF instrument (Bruker Daltonics). For acquisition of the mass spectra, 100 laser shots were

typically applied at several positions. Only positively charged compounds were analyzed, and ∼300 single-shot spectra were accumulated to obtain a good signal-to-noise ratio. Spectra were recorded in reflector mode for peptides and colored compound and in linear mode for proteins. Calibration was done externally. The Flex Analysis version 2.4 software packages provided by the manufacturer were used for data processing. Glass plates were scanned for fluorodensitometry using Fluoro-S imager, and band intensity was quantified using Quantity One software (both Bio-Rad Laboratories). RESULTS AND DISCUSSION Preparation of Monolithic Thin Layers. Based on our previous successful work with poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic columns for the reversed-phase separation of proteins in capillary columns prepared via photoinitiated Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

491

polymerization,35 we decided to use this polymer also in the present study. UV light-initiated photopolymerization has a number of advantages such as high speed and operation at room temperature.36,37 In addition, polymerization is restricted to the irradiated areas while monomers do not convert to polymer in those areas that are not illuminated. Obviously, UV transparent “molds”, monomers, and porogens have to be used to achieve the desired photopolymerization. In the past, a variety of polymer particles including cellulose, chitosan, polyolefins, and poly(styrene-co-divinylbenzene) have been used for the preparation of TLC plates.38-41 For the sake of comparison with our experimental results, we prepared a monolithic layer from a mixture described by Premsteller et al.42 consisting of styrene, divinylbenzene, 1-decanol, and tetrahydrofuran with azobisisobutyronitrile as an initiator. However, the polymerization of these UV-absorbing monomers required thermal initiation, which was carried out at 70 °C. In addition to the extensive 24-h polymerization time required for this type initiations compared to the only 15-min-long photoinitiated polymerizations the poly(styrene-co-divinylbenzene) layers were poor in quality and several defects such as cracks and nonuniform thickness could be observed. Therefore, this polymer was abandoned and most of the work was carried out using poly(butyl methacrylateco-ethylene dimethacrylate) layers prepared via photoinitiated polymerization. Our polymerizations were carried out in a simple mold formed by two glass plates and a Teflon film gasket. Since one of the plates serves as backing for the thin layer, the monolith must adhere to its surface. This is easily achieved by functionalization of its surface with 3-(trimethoxysilyl)propyl methacrylate. These pendent methacrylate moieties are then incorporated into the monolithic structure during the polymerization process and covalently attach the monolith to the glass surface. In contrast, the other glass plate is removed after the polymerization process is completed. Therefore, this cover plate should not interact with the polymer layer. We used nonfunctionalized glass plates and did not observe any significant damage of surface of the monolithic thin layer after the cover plate was removed. Alternatively, this glass plate can be functionalized with fluorinated silane to avoid sticking. Micrographs shown in Figure 1 demonstrate the typical globular structure of the 150-µm monolithic layer and its adherence to the functionalized glass plate. TLC Separation of Colored Compounds. Proof of concept was first demonstrated with a separation of two colored compounds, methylene blue and methyl red. Figure 2 shows separation of this binary mixture that was achieved in 5 min using the mobile phase consisting of a 6:4:3 ethyl acetate-ethanol-water mixture. The separation was repeated five times using different plates. Each sample was spotted five times on the monolithic layer. The relative standard deviation (RSD) for the Rf values was found to be 2.25 and 2.44% for methylene blue and methyl red, (35) Lee, D.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 2004, 1051, 53-60. (36) Yu, C.; Svec, F.; Fre´chet, J. M. J. Electrophoresis 2000, 21, 120-7. (37) Yu, C.; Xu, M.; Svec, F.; Fre´chet, J. M. J. J. Polym. Sci. Pt. A, Polym. Chem. 2002, 40, 755-69. (38) Okumura, T. J. Chromatogr. 1980, 184, 37-78. (39) Gocan, S. J. Chromatogr. Sci. 2002, 40, 538-49. (40) Aboul-Enein, H. Y.; El-Awady, M. I.; Heard, C. M. Pharmazie 2002, 57, 169-71. (41) Remcho, V. T.; Tan, Z. J. Anal. Chem. 1999, 71, 248A-55A.

492

Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

respectively. The plate with separated compounds was then dried and the top spot of methylene blue subjected to MALDI-TOF mass spectrometry. Figure 2 also shows the mass spectrum with the expected peak at m/z ) 284.044. It is worth noting that no matrix has been used for this molar mass determination since its peaks would interfere with the analyzed compound. In this specific case, ionization was achieved directly from the surface of the monolithic polymer in a way similar to desorption and ionization of other compounds enhanced by the surface of the monolith that we reported elsewhere.43 TLC Separation of Peptides. In contrast to the colored compounds above, standard peptides cannot be observed in both visible and UV light. Therefore, we labeled them with fluorescamine before spotting on the layer prepared on the MALDI target plate. The plate was then developed in a saturated chamber containing a solution of TFA in acetonitrile-water mixture. Clearly, this solution is very similar to the mobile phase used for the separation of similar peptides in HPLC mode.35 Migration to the distance of 6 cm requires 5-6 min using 0.1 vol % TFA in 40 vol % aqueous ACN as the mobile phase. The location of the separated peptides was first detected visually using a UV lamp. Figure 3 shows a good separation of all three spotted peptides. The Rf values were found to be 0.52, 0.41, and 0.30 for [Sar1,Ile8]angiotensin II, angiotensin II, and neurotensin, respectively. Good repeatability was again demonstrated with five different plates and doubled spotting of mixtures affording RSD of the Rf values 2.9, 1.8, and 1.9%, respectively. Although a spot can be identified from its Rf value, MALDITOF MS analysis facilitates characterization without additional separation experiments. Since the direct ionization of peptides from the monolithic layer was very poor, R-cyano-4-hydroxycinnamic acid was combined with the spot to enhance ionization, leading to a significant appreciation of ionization efficiency. It is also likely that the matrix solution served as an extractant in a manner that increased the concentration of the peptide at the top of the monolithic layer as suggested by Gusev.3 Both spraying and spotting protocols were tested for matrix application. The former was preferred since lateral migration of compounds in the thin layer was observed for the latter after the droplet of matrix solution was applied. This migration then moved the spot away from the original location and led to a decrease in resolution. MALDI-TOF MS spectra of 250 fmol of [Sar1,Ile8]-angiotensin II, angiotensin II, and neurotensin also shown in Figure 3 indicate that the spots contain both the original peptides and their fluorescamine-labeled counterparts. For example, the top MALDI spectrum contains peaks for both [Sar1,Ile8]-angiotensin II with m/z ) 970.597 and the same peptide fluorescently labeled with fluorescamine having m/z ) 1159.944. TLC Separation of Proteins. The TLC performance of the monolithic polymer layer was further tested using a standard protein mixture containing four proteins, insulin, cytochrom c, lysozyme, and myoglobin. In the first series of experiments, these proteins were labeled with fluorescamine before spotting on the monolithic plates. Development was optimized using different mobile phases comprising acetonitrile or methanol in aqueous 0.1 (42) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 438693. (43) Peterson, D. S.; Luo, Q.; Hilder, E. F.; Svec, F.; Fre´chet, J. M. J. Rapid Commun. Mass Spectrom. 2004, 18, 1504-12.

vol % TFA. The best solvent system for the protein mixture included 0.1% TFA in 55 vol % aqueous acetonitrile. After separation, the plates were scanned by fluorodensitometer at 360 nm. Figure 4A shows the separation of each protein spotted in a 1 pmol/µL solution. This separation was then repeated on a monolithic layer attached to the MALDI target, and the spots were analyzed with MALDI-TOF MS. Typically, 0.5-1 µL of the solution containing 0.5-1 pmol of the protein was loaded. Panels B and C in Figure 4 show the MS spectra of fluorescently labeled insulin and myoglobin, respectively. Again, both labeled and unlabeled species can be observed. Test of repeatability of the retention factors Rf for proteins was carried out again with five different plates. Table 1 summarizes the repeatability of the retention factor. Finally, a solution containing 400 fmol of unlabeled cytochrome c, lysozyme, and myoglobin was loaded on the monolith layer attached to stainless steel MALDI target. After development, the plates were dried and again analyzed using MALDI-TOF MS using sinapinic acid as a matrix sprayed over the plate. Sinapinic acid was used in this experiment since it is considered to be the matrix of choice for proteins in a wide mass range. Figure 5 shows the MALDI spectra of each separated protein. The detection of nonlabeled proteins was achieved by marking the start point before development and scanning along the developed line. In the future, we plan to use the Flex_imaging software (Bruker Daltonics) for automated detection. CONCLUSIONS This preliminary study demonstrates that a layer of a porous polymer monolith conveniently obtained by photopolymerization can be used for the separation of small molecules, peptides, and proteins in TLC mode followed by laser desorption/ionization mass spectrometric detection. We believe that further optimization of both the chemistry of the monolithic polymer and its porous properties will enable separations of even more complex samples followed by matrix-free MALDI-TOF-MS. We are currently explor-

ing the preparation of such thin monolithic layers directly on the MALDI target plate, which simplifies transfer of the layer with separated compounds in the mass spectrometer. Since TLC is easy to implement in 2-D format, we are also studying the combination of 2-D TLC with MALDI MS in order to significantly increase the “peak capacity” of the plate. Another part of this project concerns further decrease in the thickness of the layer. Since Teflon films 5-10 µm thin are difficult to handle, we are exploring the use of photolithographic techniques to obtain gasketlike structures with the desired thin thickness. We also synthesize new monomers for the preparation of monolithic layers containing chemistries similar to those of typical MALDI matrixes in order to enhance efficiency of ionization and eliminate the need to apply a low molecular weight matrix. Since the chemistry of the monolithic layer can be varied in a very broad range by incorporating monomers with numerous functionalities, layers with controlled affinity toward specific compounds are readily available thereby further increasing the applicability and versatility of this technique. Preliminary results with monolithic thin layers presented in this report clearly demonstrate the feasibility of our approach and open new avenues facilitating rapid separations of complex samples using very simple means. ACKNOWLEDGMENT Support of this work by the Austrian Genome Program (GENAU) and West Austrian Initiative for Nano Networking (MVITWINN) is gratefully acknowledged. Work at the Molecular Foundry was supported by the Director, Office of Science, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, of the U.S. Department of Energy under Contract DE-AC02-05CH11231. Received for review August 16, 2006. Accepted October 13, 2006. AC061527I

Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

493