Surface-Alkylated Polystyrene Monolithic Columns for Peptide

Mariola Batycka , Neil F. Inglis , Ken Cook , Alex Adam , Douglas Fraser-Pitt , David G. E. Smith , Laura Main , Anneke Lubben , Benedikt M. Kessler...
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Anal. Chem. 2002, 74, 2336-2344

Surface-Alkylated Polystyrene Monolithic Columns for Peptide Analysis in Capillary Liquid Chromatography-Electrospray Ionization Mass Spectrometry Xian Huang,* Sheng Zhang, Gary A. Schultz, and Jack Henion

Advion BioSciences, Inc., 30 Brown Road, Ithaca, New York 14850

Macroporous poly(styrene-divinylbenzene) (PS-DVB) monoliths were prepared by in situ polymerization in PEEK, fused silica, or stainless steel tubing having an inner diameter of 75 or 125 µm. A process is described for subsequent alkylation of the flow-contacting surfaces of the monoliths. The process treats all the surfaces including through-pore surfaces of the rigid macroporous monolith with a solution containing a dissolved FriedelCrafts catalyst, an alkyl halide (1-chlorooctadecane), and an organic solvent. This process produces an improved reversed-phase liquid chromatographic separation of peptides compared to an unmodified monolithic PS-DVB column. The surface octadecylation is not necessary for a reversed-phase separation of proteins since both unmodified and modified columns provide comparable results. Tryptic protein digests, standard proteins, and standard peptides were used to evaluate the monolithic columns by employing electrospray mass spectrometry detection. Potential applications in proteomics studies by mass spectrometry, which use the alkylated monolithic column engaged onto the nanofabricated electrospray ionization chip, are also discussed. Capillary reversed-phase liquid chromatography (LC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS) is an important tool for the analysis of complex peptide mixtures from proteolytic digests.1,2 Since such applications are often sample-limited, miniaturization of the LC column inner diameter becomes more important to enhance the concentration-sensitive detection by sample preconcentration.3-5 With miniaturized LC providing its low volumetric flow rate, ionization efficiency of electrospray for MS detection tends to be increased. Recently, making the packing material in a monolithic format suggests a * Corresponding author. Phone: (607) 257-0183. Fax: (607) 257-0359. E-mail: [email protected]. (1) Wehr, T. LCGC North Am. 2001, 19, 702-711. (2) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242247. (3) Raymackers, J.; Daniels, A.; De Brabandere, V.; Missiaen, C.; Dauwe, M.; Verhaert, P.; Vanmechelen, E.; Meheus, L. Electrophoresis 2000, 21, 22662283. (4) Vissers, J. P. J. Chromatogr., A 1999, 856, 117-143. (5) Oosterkamp, A. J.; Gelpi, E.; Abian, J. J. Mass Spectrom. 1998, 33, 976983.

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new trend for capillary columns.6-8 Such capillary columns usually consist of one piece of polymer-based or silica-based monoliths having flow-through pores, and they have been recently applied successfully for capillary electrochromatography (CEC).7,9-16 The key advantages provided by monolithic columns include the absence of frits to retain the packed bed, a stable chromatographic bed that cannot develop voids, adjustable bed porosity or pore diameter, and possible low column back pressure under higher mobile-phase flows.10,12 Although silica-based monolithic columns made by sol-gel technology have also become attractive in CEC,7,15,16 polymerbased monolithic columns have been widely accepted due to the simplicity and convenience of their preparation, the versatility of their surface chemistry, and their chemical stability over the pH range of 1-14. The synthetic polymer, poly(styrene-divinylbenzene) (PS-DVB), which has served as HPLC stationary-phase supports (particles) for many years,17-19 is also a good candidate as the stationary-phase support material of a monolithic column.12,20,21 In some cases, the PS-DVB surface can be used as a reversedphase liquid chromatographic stationary phase without further modification since it is highly hydrophobic. For example, PS-DVB particles were found very effective for the rapid analysis of proteins (6) Svec, F.; Peters, E. C.; Sykora, D.; Frechet, J. M. J. Chromatogr., A 2000, 887, 3-29. (7) Tanaka, N.; Kobayashi, H.; Nakanishi, K.; Minakuchi, H.; Ishizuka, N. Anal. Chem. 2001, 73, 420A-429A. (8) Majors, R. E. LCGC North Am. 2001, 19, 1021-1104. (9) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. Anal. Chem. 1997, 69, 3646-3649. (10) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. Anal. Chem. 1998, 70, 2288-2295. (11) Huang, X.; Zhang, J.; Horva´th, C. J. Chromatogr., A 1999, 858, 91-101. (12) Gusev, I.; Huang, X.; Horva´th, C. J. Chromatogr., A 1999, 855, 273-290. (13) Zhang, S.; Huang, X.; Zhang, J.; Horva´th, C. J. Chromatogr., A 2000, 887, 465-477. (14) Asiaie, R.; Huang, X.; Farnan, D.; Horva´th, C. J. Chromatogr., A 1998, 806, 251-263. (15) Pursch, M.; Sander, L. C. J. Chromatogr., A 2000, 887, 313-326. (16) Hayes, J. D.; Malik, A. Anal. Chem. 2000, 72, 4090-4099. (17) Rounds, M. A.; Regnier, F. E. J. Chromatogr. 1988, 443, 73-83. (18) Maa, Y. H.; Lin, S.; Horva´th, C.; Yang, U.; Crothers, D. M. J. Chromatogr. 1990, 508, 61-73. (19) Mohammad, J.; Jaderlund, B.; Lindblom, H. J. Chromatogr., A 1999, 852, 255-259. (20) Wang, Q. C.; Svec, F.; Frechet, J. M. Anal. Chem. 1993, 65, 2243-2248. (21) Petro, M.; Svec, F.; Frechet, J. M. J. Chromatogr., A 1996, 752, 59-66. 10.1021/ac011202w CCC: $22.00

© 2002 American Chemical Society Published on Web 04/17/2002

in reversed-phase HPLC mode.18,22 However, for separation and identification of much smaller biomolecules such as proteolytic peptides, the unfunctionalized PS-DVB particles usually provide relatively poor chromatographic resolution.23,24 It has been shown by Huber et al.23 that alkylation of highly cross-linked PS-DVB particles to graft octadecyl (C-18) chains on their surfaces is necessary to achieve good resolution for reversed-phase LC of peptides. For separation of oligonucleotides in reversed-phase LC, alkylation of PS-DVB particles was also found necessary by Huber et al.24 The reversed-phase LC determination of peptides on unmodified PS-DVB monoliths prepared with different porogenic solvents and conditions has not been thoroughly investigated. Similar to the PS-DVB particles, an unmodified PS-DVB monolith may also need the surface alkylation for better resolutions of peptides, although some acceptable reversed-phase separations of peptides on unmodified PS-DVB columns have been reported recently.25 It would appear worth determining whether the surface alkylation of a PS-DVB monolith helps to improve peptide separation in a reversed-phase LC mode, especially when the separation on the unmodified monolith is not acceptable. As an option, alkyl groups can be directly imparted from a comonomer such as alkylstyrene or even other non-styrene comonomers having alkyl chains. In this case, however, finding a suitable porogen that dissolves monomers having long alkyl chains and also precipitates the in situ-formed polymer is difficult. Furthermore, the mechanical and chemical stability of these monoliths is compromised if the alkyl chains are unnecessarily involved in the body of the polymer support. Only those alkyl groups bonded onto the polymer surface contribute to the chromatographic separation. Thus, a postpolymerization surface alkylation is preferred. In this study, the flow-contacting surfaces of a macroporous polystyrene monolith were octadecylated using a solution containing a strong Friedel-Crafts catalyst. The improved chromatographic resolution of peptides using the octadecylated monolithic PS-DVB columns coupled with nanoscale ESI-MS analysis is demonstrated in this report. EXPERIMENTAL SECTION Materials. Polyetheretherketone (PEEK) tubing (5 ft in length) and stainless steel tubing (precut, 10 cm in length) with 125-µm i.d. and 1/16-in. o.d. were purchased from Upchurch Scientific (Oak Harbor, WA). Fused-silica capillary tubing with a 20-µm-thick standard polyimide outer coating, having a 75-µm i.d. and 375-µm o.d. was purchased from Polymicro Technologies (Phoenix, AZ). Styrene (99+%), divinylbenzene (80%, mixture of isomers including 3- and 4-ethylvinylbenzene), 1-propanol (99.5%, HPLC grade), formamide (98%), 2,2′-azobisisobutyronitrile (AIBN) (98%), 3-(trimethoxysilyl)propyl methacrylate, 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), N,N-dimethylformamide (DMF) (22) Zhelev, N. Z.; Barratt, M. J.; Mahadevan, L. C. J. Chromatogr., A 1997, 763, 65-70. (23) Huber, C. G.; Kleindienst, G.; Bonn, G. K. Chromatographia 1997, 44, 438448. (24) Huber, C. G.; Oefner, P. J.; Bonn, G. K. Anal. Biochem. 1993, 212, 351358. (25) Premstaller, A.; Oberacher, H.; Walcher, W.; Timperio, A. M.; Zolla, L.; Chervet, J. P.; Cavusoglu, N.; van Dorsselaer, A.; Huber, C. G. Anal. Chem. 2001, 73, 2390-2396.

(99.8%), 1-chlorooctadecane (96%), aluminum chloride (99.99%), and nitrobenzene (99+%) were purchased from Sigma-Aldrich (Milwaukee, WI). Acetonitrile (HPLC gradient grade) was obtained from AlliedSignal (Muskegon, MI). Styrene and DVB were washed with 10% (w/v) aqueous sodium hydroxide to remove the inhibitors. The other materials were used without further purification. Water was purified and deionized with a Milli-Q system from Millipore (Bedford, MA). Sample Preparation. Standard proteins and peptides were all from Sigma (St. Louis, MO). Six proteins were mixed in water to form a solution containing 10 pmol/µL ribonuclease A (bovine), 3 pmol/µL cytochrome c (horse heart), 10 pmol/µL lysozyme (chicken), 2 pmol/µL myoglobin (horse heart), 2 pmol/µL bovine serum albumin (BSA), and 10 pmol/µL ovalbumin (chicken). Seven peptides including methionine enkephalin, leucine enkephalin, oxytocin, bradykinin, angiotensin I, angiotensin II, and luteinizing hormone releasing hormone (LH-RH) were mixed in water to form a solution containing 14 pmol/µL of each peptide. Trypsin was obtained from Promega (Madison, WI). For digestion of cytochrome c, 10 mg of cytochrome c was dissolved in a 1.0-mL solution containing 7.0 M urea, 2.0 mM dithiothreitol (DTT), and 50 mM Tris-HCl, pH 8.0. The mixture was incubated at 85 °C for 45 min before it was diluted by 1:10 in 50 mM ammonium bicarbonate, pH 7.8, to form a solution containing 1 mg/mL denatured protein. Trypsin was then added to the solution at an enzyme-to-substrate ratio of 1:25 (w/w). The digestion was performed at 37 °C for 16 h and stopped by the addition of 0.1% (v/v) acetic acid. For digestion of myoglobin with guanidine, the procedure was the same except using 6.0 M guanidine hydrochloride instead of 7.0 M urea. The digests were stored at -20 °C ready for use. Instrumentation. In the LC-ESI-MS system for evaluating a monolithic capillary column, a microgradient syringe pumping system (MicroPro, Eldex Laboratories, Napa, CA) was used to deliver mobile phase to a connected capillary column. In the mobile-phase flow line, a microelectric two-position valve actuator (Valco Instruments, Houston, TX) with 1-µL injection volume was connected after the pump. A high-pressure capillary graduated microsplitter valve (Upchurch Scientific) was connected after the sample injector and before the column inlet, which typically split 1/ 100 of the main flow to the column while the remainder was directed to waste. All connection capillaries were fused-silica capillaries. The mobile-phase flow rate before the split was typically maintained at 30 µL/min, while the postsplit flow rate for the column was maintained at 200-300 nL/min. The column outlet was typically connected to a tapered fused-silica capillary for nanoelectrospray (tip end i.d. 10 µm, flame-pulled from a fusedsilica capillary having 150-µm o.d. and 50-µm i.d.). For the chip-based electrospray, only the 10-cm-long conductive stainless steel tubing containing the monolithic packing was used since the tubing must be conductive for the connection of high voltage.26 One of its ends was further machined on the metal to form a convex shape so that an O-ring was fitted on. A microfabricated silicon chip with an array of nanospray nozzles was used as the nanoelectrospray ionization device. The system for holding (26) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063.

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Figure 1. Use of the screw-top glass vial having an open-top cap with a septum: (a) Purging or bubbling with helium. (b) Filling the empty capillary column with the solution. (c) Sealing the solutionfilled column by inserting the other end into the vial. (d) Monolithic matrix formed in both the capillary tubing and the vial after the polymerization.

and moving the chip on which the column outlet (with the O-ring) was engaged was described in a previous publication.26 A LCT time-of-flight mass spectrometer (Micromass, Beverly, MA) running MassLynx NT software was used. Mass spectra were acquired in the range of m/z 700-3000 for proteins and m/z 3801700 for peptides using 2- (for proteins) or 1-s (for peptides) ion integration times. Monolithic Column Preparation. Prior to their surface alkylation, the monolithic capillary columns were prepared by in situ formation of a macroporous PS-DVB monolith in the PEEK, stainless steel, and fused-silica tubing. The 125-µm-i.d. PEEK tubing having a length ∼125 cm was directly used as the mold for the in situ polymerization without inner surface pretreatment. Each end of the PEEK tubing was connected with a 10-cm-long standard fused-silica capillary (75-µm i.d.) by using a connection union from Upchurch Scientific. A screw-top glass vial (1.5 mL) having an open-top cap with a Teflon-faced plastic septum was used for loading the initial polymerizing solution into the PEEK tubing. A 1-mL solution, containing 20% (v/v) styrene and 20% (v/v) DVB as the monomers, 40% (v/v) 1-propanol and 20% (v/v) formamide as the porogen, and 0.3% (w/v) AIBN as the initiator, was prepared and filtered through a cellulose membrane having 0.22-µm pores (Millipore). The filtrate was placed into the screw-top vial, and the vial was then closed with the cap. The PEEK tubing was connected to the vial by inserting one of its fused-silica capillary ends into the vial (but above the liquid level) through the septum. A fused-silica capillary connected to a helium source of 50 psi (0.34 MPa; 1 psi ) 6894.76 Pa) was also inserted into the vial (to the bottom). The solution was purged this way with helium for 15 min (Figure 1a). Subsequently, the capillary end of the helium source was pulled back to a position above the liquid level in the vial, while the capillary end of the PEEK tubing was inserted deeper and immersed in the liquid. The solution contained in the vial was then pressurized and forced into the PEEK tubing (Figure 2338

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1b). The helium source capillary was pulled out from the vial after the PEEK tubing was overfilled with the solution. With the liquid running, the other extended end of the PEEK tubing was quickly inserted through the septum to the bottom of the vial to ensure the absence of bubbles in the liquid path. The storage vial then served to keep the liquid filled in the capillary path (Figure 1c). The vial with the tubing were placed in an oven and heated at 65 °C for 24 h, and the monolithic porous matrix was formed in both the vial and the capillary (Figure 1d). After the vial was pulled from the capillary, the residual mixture (mostly the porogen) was removed from the tubing by nitrogen pressure. The 125-cm-long PEEK tubing containing the porous PS-DVB monolith was cut into four pieces of ∼31 cm each in length. These monoliths were thoroughly washed with methylene chloride (2 µL/min for 1 h) and DMF (2 µL/min for 2 h). The precut 125 µm i.d. × 10 cm stainless steel tubing was washed with methanol and blown dry with nitrogen for 1 h at 80 psi inlet pressure. Each end of the tubing was connected with a 10-cm-long standard fused-silica capillary (75-µm i.d.) by using a connection union from Upchurch Scientific. The subsequent procedure to prepare a PS-DVB monolith in the stainless steel tubing is the same as that for PEEK tubing. A PS-DVB monolith covalently bonded in a fused-silica capillary tubing was also prepared prior to its surface alkylation. The untreated fused-silica capillary (75-µm i.d., 375-µm o.d., and 90 cm in length) was first silanized by the method of Huang and Horva´th27 so that its inner wall was enriched with bonded vinyl groups. The bifunctional agent 3-(trimethoxysilyl)propyl methacrylate and the inhibitor DPPH were used for the silanization. The procedures for the in situ polymerization and the postpolymerization wash were the same as described above. The 90-cmlong capillary containing the porous PS-DVB monolith was cut into three pieces each of 30 cm in length after the polymerization and before the wash. Pore Size Distribution Test. Mercury intrusion porosimetry (MIP) was used for measuring the pore size distribution of the porous PS-DVB monolith. BET nitrogen adsorption was used as an additional test, which is the standard measurement giving specific surface area and information about smaller (nanometersized) pores. Since the quantity of the porous material in a capillary tubing was too small, directly testing it by MIP and BET methods was technically difficult. Instead, the monolithic PS-DVB block (typically ∼0.4 g) formed in the small glass vial (shown in Figure 1d) was tested by MIP to represent the feature of the monolith simultaneously formed in a capillary. We believe that the PS-DVB monolith formed in the same batch should be comparable in their porous structures, since the polymerization solution and the polymerization conditions were the same. By cutting away the top part of the glass vial, the monolithic PS-DVB block was carefully taken out and put in a 50-mL beaker. The monolithic block was then immersed and infused thoroughly by methylene chloride and acetonitrile, respectively. After the wash, the monolithic block was dried at 65 °C for 6 h in an oven with a nitrogen purge before the MIP or BET tests. The MIP and BET tests were conducted by Porous Materials, Inc. (Ithaca, NY). Octadecylation of a PS-DVB Monolith. Both monolithic capillary columns prepared above were used for the surface (27) Huang, X.; Horva´th, C. J. Chromatogr., A 1997, 788, 155-164.

alkylation to form a stationary phase of hydrocarbons containing 18 carbon atoms. Thus, we refer to the alkylation as octadecylation. The method for octadecylating a PEEK capillary column, a stainless steel capillary column, or a fused-silica capillary column containing a PS-DVB monolith was the same. Similar to the method used above for forcing a solution into a capillary, each end of a monolithic column was extended with a fused-silica capillary using a connecting union. A screw-top glass vial (1.5 mL) having an open-top cap with a Teflon-faced plastic septum was also used for delivering the C-18 alkylating reagent into a monolithic column. Typically, a liquid contained in the vial was pressurized by a nitrogen source of 80 psi (0.55 MPa) introduced with a fused-silica capillary inserted through the septum. With one end inserted into the capped vial containing 1.0 mL of nitrobenzene, a monolithic column was first rinsed with nitrobenzene for 1 h before the octadecylation. A total of 25 mg of aluminum chloride powder was put into another screw-top glass vial, and 0.5 mL of nitrobenzene was added. After stirring for a few minutes, 0.5 mL of 1-chlorooctadecane (liquid) was added. A uniform solution was formed after further stirring. The vial was then closed using the open-top cap with the septum. The nitrobenzene-washed monolithic column was purged with the prepared solution for ∼1 h at its inlet pressure of 80 psi. The column was then sealed at both ends and placed in an oven at 60 °C for 12 h. The postoctadecylation reaction mixture was purged from the alkylated monolith by pressurized nitrobenzene. The octadecylated monoliths were washed sequentially with 0.3 mL of N,N-dimethylformamide, 0.3 mL of 1 M HCl aqueous solution, 0.3 mL of water, and 0.5 mL of acetonitrile, pressurized with 80 psi nitrogen. Then the capillaries were neatly cut into shorter pieces as the resulting capillary monolithic columns having certain lengths. RESULTS AND DISCUSSION Physical Features of a PS-DVB Monolith. The covalent bonding between a polymer monolith and the inner wall of a fusedsilica capillary is essential for mechanical stability of the monolith. A monolith without covalent bonding onto a fused-silica capillary inner wall is not mechanically stable and tends to be pushed out of the capillary at a normal flow pressure.12 However, for a polymer monolith molded in PEEK tubing prepared from the procedure described above (without covalent bonding), we found that the monolith was held in the PEEK tubing strongly enough to withstand a head pressure as high as 200 bar delivered by a continuous water flow. To find out the reason, we removed several short segments (about 1-3 mm in length) of the PS-DVB monoliths from different PEEK tubings by a mechanical way and observed them using a scanning electron microscope. Surprisingly, unlike a PS-DVB monolith formed in a bare fused-silica capillary (without covalent bonding)12 but more like a PS-DVB monolith in a vinyl-functionalized fused-silica capillary (with covalent bonding),11,12 the monolith in a PEEK tubing has a dense (almost nonporous) thin annular outer layer firmly attached on the PEEK wall. It indicates the enhanced precipitation and attachment of the polymer globules onto the PEEK inner wall during some early stage of the in situ polymerization due to the much higher hydrophobicity or compatibility of PEEK surface compared with the hydrophilic and rigged fused-silica surface. In addition, PS-DVB monoliths in situ formed in the 125 µm i.d. ×

Figure 2. Pore size distribution of a PS-DVB monolith as measured by mercury intrusion porosimetry.

10 cm stainless steel tubing were also tested and proven to be able to withstand a head pressure as high as 200 bar delivered by a continuous water flow. The high mechanical stability of the monolith held by such metal tubing is most likely due to the high surface roughness of the tubing inner wall. It should be noted that the reversed-phase liquid chromatographic separations in this study were affected by the monolithic packing, not the tubing material. The effect of the material type of the holding tubing on the separations proved to be not noticeable based on our experiments. Use of the different tubings (PEEK, stainless steel, and the pretreated fused silica) in this study was to determine their feasibility as viable supports. The hydrodynamic properties and the surface area of the flowthrough polymeric monolith are related to its porous structure configuration. Under the experimental conditions in this study using 40% (v/v) 1-propanol and 20% (v/v) formamide as the porogen, the resulting capillary monolith consisted of throughpores equivalent to cylindrical pores with a diameter of 4 µm as shown in Figure 2. Such relatively large through-pore size for the monolith presents an advantage in terms of maintaining a suitable flow velocity under much lower head pressure, compared with a packed bed with 3-5-µm-sized particles. The monolithic columns in this study were operated at the typical pressure drop of ∼10 psi/cm. However, the low specific surface area (less than 10 m2/g) measured by multipoint BET nitrogen adsorption/desorption for the porous polymer monolith indicates that in the walls of its micrometer-sized pores there was an absence of nanometer-sized (e.g., 10-100 nm) pores. Thus, the monoliths presented a single modal pore size distribution consisting of 4-µm-sized pores. Porosity of the monolith was estimated as 0.60 (v/v). Uniform Solution for the Surface Treatment. The FriedelCrafts reaction28 was adopted for the surface alkylation of the PSDVB monoliths. The process comprised an important step of treating the pore surfaces by filling the monolith with a uniform solution containing the Friedel-Crafts catalyst, an alkyl halide, and an organic solvent. It is essential that the Friedel-Crafts catalyst and the alkyl halide are dissolved in the selected solvent (28) Roberts, R. M.; Khalaf, A. A. Friedel-Crafts Alkylation Chemistry; Marcel Dekker: New York, 1984.

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so that a uniform solution can be formed. By maintaining all reactants and byproducts in solution, clogging and precipitation were eliminated during the process including the subsequent washing to remove the postalkylation reaction product mixture from the functionalized PS-DVB monolith. Theoretically, a strongly catalyzed Friedel-Crafts reaction on a free benzene ring is very fast even under very low-temperature conditions (lower than 0 °C).28 However, the temperature for the monolith surface octadecylation in this study was maintained higher than room temperature (60 °C) to overcome the steric resistance of the polymer surface and to enhance the diffusion to the polymer surface. PS-DVB particles can be octadecylated by Friedel-Crafts reaction as reported by Huber et al.24 In that process, solid aluminum chloride as the catalyst was directly added to a suspension of nonporous PS-DVB particles in an alkyl chloride (1-chlorooctadecane). No other solvent was added during the multiphase reaction. This procedure does not appear suitable for alkylating a porous polystyrenic monolith. First, a solid-state catalyst is difficult to introduce into the pores or through-channels of a monolith if the catalyst is not soluble in the alkylating solution. Second, the pores or channels of a monolith could become clogged if any solid material precipitated and remained inside the monolith during the Friedel-Crafts reaction. Although there are a few available liquid-state Friedel-Crafts catalysts, most of them are not suitable for the application described here. For example, tin(IV) chloride is a liquid and can flow into the monolith porous structure. However, insoluble substances produced during the Friedel-Crafts reaction may occur, plus it is a very weak alkylation catalyst as well. These problems were avoided using the solutionbased procedure described in this study. Influence of Surface Octadecylation on Separation of Proteins and Peptides. It was reconfirmed by the associated experiments in this study that the unmodified PS-DVB surface offered good separation of proteins and the surface octadecylation of the PS-DVB monoliths was not necessary for protein separation under gradient reversed-phase LC-ESI-MS conditions. The mixture of six Sigma standard proteins were baseline separated with good peak shapes using both unmodified and octadecylated PS-DVB monolith columns as shown in Figure 3, which coincides with the protein separations on unmodified and octadecylated PSDVB beads carried out by Huber et al.23 The PS-DVB monoliths with and without surface octadecylation provided similar reversedphase chromatographic separations for proteins. However, separation of peptides with the unmodified PS-DVB monolith was found not as good as that with the octadecylated PS-DVB monolith. The results shown below demonstrate the importance of the surface alkylation of a PS-DVB monolith for improved peptide separations. The reversed-phase LC behavior of peptides was investigated by analyzing a peptide mixture from cytochrome c tryptic digestion on both unmodified (Figure 4) and octadecylated (Figure 5) capillary PS-DVB monolithic columns. In both Figure 4 and Figure 5, the results are presented by the respective total ion chromatograms (TICs) and the extracted ion chromatograms corresponding to individual peptides. In Figure 4, the selected extracted ion chromatograms for the four tryptic fragments T2, T6, T3, and T1 display no separation although the peaks are sharp and with good shapes. The peaks T9, T7, and T12 in Figure 4 are broad and 2340 Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

Figure 3. Comparison of reversed-phase LC separation of standard proteins represented by the total ion (TICs) chromatograms on the PS-DVB monolithic column before and after the surface octadecylation. Column: 125-µm i.d., 1/16-in. (1.575 mm) o.d., and length 10cm PEEK tubing containing the macroporous PS-DVB monolith. Sample mixture introduced into the column: 100 fmol of ribonuclease A (peak 1), 30 fmol of cytochrome c (peak 2), 100 fmol of lysozyme (peak 3), 20 fmol of BSA (peak 4), 20 fmol of myoglobin (peak 5), and 100 fmol of ovalbumin (peak 6). Mobile phase: A ) 0.1% (v/v) TFA in water; B ) 0.1% (v/v) TFA in acetonitrile. Gradient elution program: 0 f 1 f 15 min, 0% f 30% f 60% B. Flow rate in column: 0.3 µL/min. Temperature: 20 °C. Voltage for ESI with the fused-silica capillary tip: +3.5 kV. MS detection: 700-3000 m/z.

poorly resolved. The peak T4 in Figure 4 is separated from the others but is broad with poor shape. In Figure 5, however, peaks for the same tryptic peptides T2, T6, T3, and T1 are resolved, and the peak shapes for T4 and T9 are improved. T7 and T12 in Figure 5 are also separated, although their shapes are still poor. In fact, the poor peak shapes for T7 and T12 on both unmodified and octadecylated columns were caused partly by the ion suppression of urea which coeluted with the most hydrophilic tryptic fragments. Since the TICs shown in Figures 4 and 5 were obtained under gradient elution rather than isocratic elution, it is not appropriate to use their peak widths or peak widths at half-height to calculate the column efficiency or theoretical plate height. However, for the purpose of further comparing the column performance before and after the surface octadecylation, relative peak width at halfheight (i.e., the ratio of peak width at half-height over its retention time) is used to compare two corresponding peaks between the two cases. By its meaning, the relative peak width at half-height should represent how good the peak is for the corresponding individual analyte under gradient elution. Table 1 compares the relative peak widths at half-height which were measured from the extracted ion chromatograms in Figure 4 and Figure 5. As shown in Table 1, the relative peak widths at half-height for all tryptic fragments were generally reduced (except T3 and T6). The relative peak widths at half-height for T1, T5, T3, and T6 remained

Figure 4. Performance of the unmodified PS-DVB monolith for the separation of peptides from the tryptic digest of cytochrome c represented by the TIC chromatograms and extracted ion content chromatograms. Column: 125-µm i.d., 1/16-in. (1.575 mm) o.d., and length 10-cm PEEK tubing containing an unmodified macroporous PS-DVB monolith. Sample introduced into the column: 0.7 pmol each of the peptides from the digest. Mobile phase: A ) 0.1% (v/v) acetic acid and 0.01% (v/v) heptafluorobutyric acid in water; B ) 0.1% (v/v) acetic acid and 0.01% (v/v) heptafluorobutyric acid in acetonitrile, Gradient elution program: 0 f 1 f 10 f 15 min, 0% f 10% f 30% f 60% B. Flow rate in column: 0.3 µL/min. Temperature: 20 °C. Voltage for ESI with the tapered fused-silica capillary: +3.5 kV. MS detection: 380-1700 m/z.

Figure 5. Performance of the octadecylated PS-DVB monolith in the separation of peptides from the tryptic digest of cytochrome c represented by the TIC chromatograms and extracted ion content chromatograms. Column: 125-µm i.d., 1/16-in. (1.575 mm) o.d., and length 10-cm PEEK tubing containing a macroporous PS-DVB monolith with octadecylated surfaces. Sample introduced into the column: 0.7 pmol each of the peptides from the digest. Mobile phase: A ) 0.1% (v/v) acetic acid and 0.01% (v/v) heptafluorobutyric acid in water; B ) 0.1% (v/v) acetic acid and 0.01% (v/v) heptafluorobutyric acid in acetonitrile. Gradient elution program: 0 f 1 f 10 f 15 min, 0% f 10% f 30% f 60% B. Flow rate in column: 0.3 µL/min. Temperature: 20 °C. Voltage for ESI with the fused-silica capillary tip: +3.5 kV. MS detection: 380-1700 m/z.

unchanged or even slightly increased after the surface octadecylation, which was probably due to the increase in hydrophobicity after the surface alkylation. The relative peak widths at half-height of T4, T9, and T7 were significantly reduced due to the surface octadecylation. Since the increase of resolution is represented not only by the narrower peak width but also by the higher selectivity, the overall improvement of the peptide separation due to the surface octadecylation is obvious. Thus, the comparison between Figure 4 and Figure 5 suggests that the octadecylated PS-DVB monolithic column provides much better reversed-phase LC separation of peptides than the unmodified PS-DVB monolithic column under the same gradient elution conditions. Moreover, for the octadecylated monolithic column, the urea additive did not significantly suppress the peptide signals, and even the extremely hydrophilic fragments T10-12 were still detectable as shown in Figure 5.

Standard peptides displayed similar behavior, which is consistent with the results described above. For the mixture containing seven Sigma standard peptides which are methionine enkephalin, leucine enkephalin, oxytocin, bradykinin, angiotensin I, angiotensin II, and LH-RH, the unmodified PS-DVB monolithic columns gave poor resolution and only angiotensin I was separated from the others. However, improved chromatographic separation of the standard peptides with better peak shapes was obtained on the octadecylated monolith column. Figure 6 shows the online LC separation and ESI-MS detection of the seven standard peptides using the octadecylated PS-DVB monolith in a fusedsilica capillary having a length of 14 cm. All seven peptides eluted from the column within a 2-min time period and were easily identified by their corresponding extracted ion peaks. Peptide identification was further confirmed by the full-scan mass spectra as shown in Figure 7, in which the specific mass spectra of the Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

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Table 1. Relative Peak Widths at Half-Heighta for Selected Tryptic Peptides on the Monolithic PS-DVB Column (10 cm in Length) beforeb and afterc the Surface Octadecylation W1/2/tR, ×10-2

tR, min peak for peptide

before

after

before

after

T4 T1 T5 T3 T6 T2 T9 T7

5.99 4.45 5.05 4.56 4.45 4.45 1.63 1.28

7.36 7.11 5.93 5.91 5.59 5.24 4.89 1.53

7.1 2.3 3.0 2.5 1.7 1.9 9.2 18.1

4.4 2.3 3.0 2.7 2.1 1.6 3.0 2.9

a W 1/2/tR, where tR is the retention time and W1/2 the width at the half peak height.36 b Corresponding to Figure 4. c Corresponding to Figure 5.

small peptides exhibit (M + nH)n+ ions, where n is the degree of protonation.29,30 Specifically, the dominant ions for methionine enkephalin and leucine enkephalin were singly charged, and doubly charged for the other five peptides. It is known that the bonding density of octadecyl chains or hydrocarbons on silica particles used as the reversed-phase chromatographic stationary phase can be quantitatively determined by certain methods, and the partition or retention mechanism of the bonded phase has been thoroughly investigated.31,32 Unfortunately, those methods do not apply for the quantitation of alkyl chains grafted in a polystyrene base. In addition, the mechanism that the PS-DVB surface offers better reversed-phase liquid chromatographic resolution for peptides is not completely clear. In related studies using unmodified PS-DVB beads,23 it was shown that peptides (having much smaller hydrodynamic radii between 1.6 and 2.6 nm compared with proteins) were trapped in the chains of the swollen polymer surface, thus giving poor separation. In that case, it was assumed the polymer surface was not totally “smooth” but had linear and branched polymer chains resulting in a swollen polymer network. It was further proposed that octadecyl groups create an average hydrocarbon layer of 1.8nm thickness and hindered access to the micropores resulting in better separation of the small peptides. As reported by other researchers, grafting octadecyl groups on PS-DVB beads23,33,34 helps to reduce unwanted π-π-interactions between aromatic moieties of the stationary phase and aromatic peptide side chains. Despite the density of the grafted octadecyl groups and the mechanism of partition behavior between peptide molecules and the retaining groups, the results obtained in this study directly confirm that surface octadecylation improves the performance of the macroporous PS-DVB monolith columns in reversed-phase LC of peptides. It must be noted that in a recent publication25 a 200-µm-i.d. monolithic PS-DVB column is reported, which was prepared by (29) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. (30) Lee, E. D.; Henion, J. D.; Covey, T. R. J. Microcolumn Sep. 1989, 1, 14-18. (31) Sentell, K. B.; Dorsey, J. G. J. Chromatogr. 1989, 461, 193-207. (32) Hetem, M. J. J. Chemically modified silica surfaces in chromatography; J. Wiley & Sons: New York, 1993. (33) Yarowsky, I.; Aguilar, M.-I.; Hearn, M. T. W. Anal. Chem. 1995, 67, 21452153. (34) Bowers, L. D.; Pedigo, S. J. Chromatogr. 1986, 371, 243-251.

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Figure 6. TIC chromatogram and extracted ion content chromatograms from the LC-ESI-MS analysis of a synthetic mixture containing seven standard peptides on the column containing a PS-DVB-C18 monolith. Column: 75-µm i.d., 375-µm o.d., and length 14-cm fusedsilica capillary containing a macroporous PS-DVB monolith with octadecylated surfaces. Sample introduced into the column: synthetic mixture containing 0.14 pmol each of the seven Sigma standard peptides, (1) methionine enkephalin, (2) leucine enkephalin, (3) oxytocin, (4) bradykinin, (5) LH-RH, (6) angiotensin II, and (7) angiotensin I. Mobile phase: A ) 0.1% (v/v) TFA in water; B ) 0.1% (v/v) TFA in acetonitrile. Gradient elution program: 0 f 8 min, 10% f 40% B. Flow rate in column: 0.3 µL/min. Temperature: 20 °C. Voltage for ESI with the fused-silica capillary tip: +3.5 kV. MS detection, 380-1700 m/z.

in situ polymerization using a different two-solvent porogen.35 As supported by its results, surface modification was not necessary since the column gave high resolution for peptides in reversedphase separation mode. However, the pore size distribution of that unmodified PS-DVB monolith was not reported. The scanning electron microscopic pictures of the monolithic structure of that column show a monolithic structure that is very fine or detailed and the macropore sizes (channel sizes) are relatively small (much less than 1 µm). An advantage of this type of monolith is that the (35) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 43864393. (36) Horva´th, C.; Melander, W. R. In Chromatography: Fundamentals and Applications of Chromatography and Electrophotometric Methods, Part A: Fundamentals; Heftmann, E., Ed.; Elsevier: New York, 1983; pp A27-A135.

Figure 7. Mass spectra of the seven standard peptides from the corresponding chromatographic peaks shown in Figure 6.

specific surface area is high and higher separation efficiency can be expected. However, relatively high head pressure must be needed to drive a flow through such a monolithic column, although no pressure data are given in that publication. The PSDVB monolithic capillary columns prepared in this study were with large through-pores equivalent to cylindrical macropores of 4 µm and needed very low pressure (10 psi/cm) to drive a flow. The large through-pores with low specific surface area might be the reason that the underivatized PS-DVB monoliths provided poor resolution for separation of peptides. The low head pressure and pressure drop not only eliminate bubbles in the hydroorganic flow line (e.g., in the dead volume between a column end and its engaged chip reservoir26) but also potentially simplify the nanoscale liquid delivery system and automation when coupled with an electrospray ionization chip.26 The purpose of the surface alkylation is to rebuild the resolution of such a type of PS/DVB monoliths for peptides. Peptide LC-ESI-MS Analysis on Octadecylated PS-DVB Monolithic Column. LC separation of a complex peptide mixture from proteolytic digests of proteins followed by mass spectrometry analysis becomes increasingly important in today’s proteomics studies. LC-MS strategies can compliment conventional 2-D gel electrophoretic separation and may enable the detection of low abundant and membrane-associated proteins.2 The miniaturized monolithic materials for peptide separation were developed for this purpose. To further evaluate the surface-octadecylated PSDVB monolithic column and to explore its potential application for proteomics, capillary LC-ESI-MS analysis of a myoglobin tryptic digest was carried out using the stainless steel capillary column containing the octadecylated PS-DVB monolith and the ESI chip.26 To represent most protein tryptic digest samples, which are usually “dirty”, myoglobin was chosen as the target protein to be identified, since its tryptic digestion is usually completed with the

Figure 8. TIC chromatogram and extracted ion chromatograms from the nanoscale LC-ESI-MS analysis of a myoglobin tryptic digest using the column containing a PS-DVB-C18 monolith and a chipbased ESI nozzle. Column: 125-µm i.d., 1/16-in. (1.575 mm) o.d., and length 10-cm stainless steel tubing containing a macroporous PSDVB monolith with octadecylated surfaces. Sample mixture introduced into the column: 0.7 pmol each of the peptides from the digest. Mobile phase: A ) 0.1% (v/v) acetic acid and 0.01% (v/v) heptafluorobutyric acid in water; B ) 0.1% (v/v) acetic acid and 0.01% (v/v) heptafluorobutyric acid in acetonitrile. Gradient elution program: 0 f 10 f 15 min, 5% f 40% f 70% B. Flow rate in column: 0.3 µL/min. Temperature: 20 °C. Voltage for ESI with the chip-based nanospray nozzle: +1.2 kV. MS detection: 380-1700 m/z.

addition of guanidine hydrochloride to facilitate denaturing of the protein. Figure 8 shows the LC-MS chromatograms of the tryptic digest of myoglobin. Peptides from the digest mixture were well separated on the stainless steel capillary column containing the PS-DVB-C18 monolith. With guanidine hydrochloride, which can significantly suppress electrospray ion current, 10 out of 15 target fragments in the mass range m/z 380-1700 were still detected. The guanidine additive suppressed only those extremely hydrophilic fragments having little or no retention under these conditions. Although only one of the nozzles was used to achieve the above separation of the tryptic peptides, the results show feasibility for using miniaturized monolithic LC columns and the chip-based nozzle array for high-throughput desalting and separation of peptides in proteomics studies. Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

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CONCLUSIONS A simple process is described for alkylating flow-contacting surfaces of a macroporous PS-DVB monolith to produce a reversed-phase liquid chromatographic column packing. Besides the PS-DVB monoliths molded by capillaries (fused-silica capillaries, PEEK tubing, or stainless steel tubing), other PS-DVB monoliths molded by various microsized horizontal channels or vertical through-holes in silicon chips or in polymer chips should be also alkylated by this process. The monolith is cross-linked polystyrene copolymer having through-pores through which a liquid can pass. The alkylation of PS-DVB monoliths with linear C-18 alkyl groups appreciably improves the reversed-phase liquid chromatographic separation of peptides. A capillary column with the surface-octadecylated macroporous PS-DVB monolith can be used for nanoscale reversed-phase LC with low-pressure drop of

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a mobile-phase flow. The results for the LC-ESI-MS of tryptic peptides obtained using the column coupled with a chip-based nanoelectrospray nozzle array have indicated feasibility regarding high-throughput simultaneous desalting and separation of peptides for proteomics studies. ACKNOWLEDGMENT Drs. Simon J. Prosser and Thomas N. Corso are gratefully thanked for their help in providing the electrospray microchip and setting up its complete handling system.

Received for review November 20, 2001. Accepted March 14, 2002. AC011202W