Control of Selectivity via Nanochemistry: Monolithic Capillary Column

Aug 31, 2010 - Monolithic High-Performance Liquid Chromatography Columns .... to enhance the electrochromatographic separation of small molecules...
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Anal. Chem. 2010, 82, 8335–8341

Control of Selectivity via Nanochemistry: Monolithic Capillary Column Containing Hydroxyapatite Nanoparticles for Separation of Proteins and Enrichment of Phosphopeptides Jana Krenkova,† Nathan A. Lacher,‡ and Frantisek Svec*,† The Molecular Foundry, E. O. Lawrence Berkeley National Laboratory, Berkeley, California 94720, and Analytical R&D, Pfizer BioTherapeutics Pharmaceutical Sciences R&D, Chesterfield, Missouri 63017 New monolithic capillary columns with embedded commercial hydroxyapatite nanoparticles have been developed and used for protein separation and selective enrichment of phosphopeptides. The rod-shaped hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The effect of percentages of monomers and hydroxyapatite nanoparticles in the polymerization mixture on the performance of the monolithic column was explored in detail. We found that the loading capacity of the monolith is on par with other hydroxyapatite separation media. However, the speed at which these columns can be used is higher due to the fast mass transport. The function of the monolithic columns was demonstrated with the separations of a model mixture of proteins including ovalbumin, myoglobin, lysozyme, and cytochrome c as well as a monoclonal antibody and its aggregates with protein A. Selective enrichment and MALDI/MS characterization of phosphopeptides fishedout from complex peptide mixtures of ovalbumin, r-casein, and β-casein digests were also achieved using the hydroxyapatite monolith. Hydroxyapatite that has been introduced as a chromatographic medium by Tiselius1 is a crystalline form of calcium phosphate with the structural formula Ca10(PO4)6(OH)2 consisting of positively charged pairs of crystalline calcium ions (C-sites) and clusters of six negatively charged oxygen atoms associated with triplets of crystal phosphates (P-sites). The combination of these active groups enables retention by at least three distinct mechanisms: cation exchange with P-sites as well as calcium coordination and anion exchange with C-sites. The predominant mechanism depends on the separation conditions, such as mobile phase buffer composition and pH and the properties of the separated analytes. Hydroxyapatite is successfully applied to the purification and separation of a wide * Corresponding author. E-mail: [email protected]. Phone: 510 486 7064. † E. O. Lawrence Berkeley National Laboratory. ‡ Pfizer. (1) Tiselius, A.; Hje´rten, S.; Levin, O. Arch. Biochem. Biophys. 1956, 65, 132– 155. 10.1021/ac1018815  2010 American Chemical Society Published on Web 08/31/2010

range of proteins, viruses, and nucleic acids.2 Nowadays, the main applications of hydroxyapatite sorbents include the separation of different subclasses of monoclonal and polyclonal antibodies, antibody fragments, and aggregates at analytical and preparative scale. This represents an alternative method to the traditional protein A affinity chromatography, ionexchange chromatography, and size exclusion chromatography.3-5 The separation mechanism on hydroxyapatite stationary phase has been studied in detail by Gorbunoff et al.6-8 They claimed that the separation does not primarily depend on any single property of the analyzed proteins, such as molecular weight, molecular size, charge density, or isoelectric point. Therefore, hydroxyapatite chromatography is a valuable complement to the arsenal of other separation techniques. Two types of hydroxyapatite sorbents are available on the market. HA Ultrogel produced by IBF Biotechnics is a crosslinked composite based on spherical agarose beads with a size of 80-160 µm containing entrapped microcrystals of hydroxyapatite. However, current applications mostly use ceramic microbeads prepared by synthesis of nanocrystals having a hexagonal cross section that are agglomerated into beads and sintered at a high temperature.9 This ceramic hydroxyapatite stationary phase, which is manufactured by Bio-Rad Laboratories, exhibits a better stability compared to HA Ultrogel and can be used under higher pressure. However, it is only available in particle sizes ranging from 10 to 80 µm. The large size of the hydroxyapatite particles limits extension of these stationary phases into the field of micro- and nanoscale separations in capillaries, thus missing the opportunity for high throughput analysis. Currently, commercial availability of hydroxyapatite nanoparticles with a size of about 50 nm offers a new prospect for the preparation of small bore columns. Unfortunately, they are too small to be used directly as a packing for columns. (2) (3) (4) (5) (6) (7) (8) (9)

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However, their incorporation into a material with suitable porous properties might provide access to new stationary phases for hydroxyapatite-based capillary columns. Monolithic columns consisting of rigid organic polymer emerged two decades ago10,11 and have been accepted as useful stationary phases for rapid liquid chromatography.12,13 This new type of column is easily prepared in situ within the confines of the column tube and exhibits excellent hydrodynamic properties, enabling the use of high flow rates. This process affords monoliths with the wide variety of chemistries that facilitates an easy tailoring of the interactions required for a specific separation mode.14-17 Traditionally, methods controlling the surface chemistry of porous polymer monoliths described in the literature comprise the copolymerization of functional monomers,10,11,18-20 chemical modification of reactive groups of the monolith,21-23 and grafting of functional polymer chains onto the surface of the pores.24-27 An additional approach, functionalization of monoliths with nanoparticles featuring a very high surface-to-volume ratio and specific chemistry, emerged recently. Nanoparticles can be embedded into the polymer monolith supports using polymerization of their dispersion in monomers and porogens28,29 or attached to the pore surface of preformed monoliths.30-37 Although functionalized latex nanoparticles were used for the preparation of particulate column packings several decades (10) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 54, 820–822. (11) Wang, Q.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1993, 65, 2243–2248. (12) Svec, F.; Tennikova, T. B.; Deyl, Z. Monolithic materials: preparation, properties, and applications; Elsevier: Amsterdam, 2003. (13) Guiochon, G. J. Chromatogr., A 2007, 1168, 101–168. (14) Urban, J.; Jandera, P. J. Sep. Sci. 2008, 31, 2521–2540. (15) Svec, F. Electrophoresis 2009, 30, S68–S82. (16) Svec, F. J. Chromatogr., A 2010, 1217, 902–924. (17) Krenkova, J.; Svec, F. J. Sep. Sci. 2009, 32, 706–718. (18) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2288–2295. (19) Xie, S.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1998, 775, 65–72. (20) La¨mmerhofer, M.; Peters, E. C.; Yu, C.; Svec, F.; Fre´chet, J. M. J.; Lindner, W. Anal. Chem. 2000, 72, 4623–4628. (21) Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1995, 702, 89–95. (22) Xie, S.; Svec, F.; Fre´chet, J. M. J. Polym. Prepr. 1997, 38, 211–212. (23) Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3131–3139. (24) Viklund, C.; Svec, F.; Fre´chet, J. M. J.; Irgum, K. Biotechnol. Prog. 1997, 13, 597–600. (25) Viklund, C.; Irgum, K.; Svec, F.; Fre´chet, J. M. J. Macromolecules 2001, 34, 4361–4369. (26) Rohr, T.; Ogeltree, D. F.; Svec, F.; Fre´chet, J. M. J. Adv. Funct. Mater. 2003, 13, 265–270. (27) Rohr, T.; Hilder, E. F.; Donovan, J. J.; Svec, F.; Fre´chet, J. M. J. Macromolecules 2003, 36, 1677–1684. (28) Li, Y.; Chen, Y.; Xiang, R.; Ciuparu, D.; Pfefferle, L. D.; Horva´th, C.; Wilkins, J. A. Anal. Chem. 2005, 77, 1398–1406. (29) Rainer, M.; Sonderegger, H.; Bakry, R.; Huck, C. W.; Morandell, S.; Huber, L. A.; Gjerde, D. T.; Bonn, G. K. Proteomics 2008, 8, 4593–4602. (30) Hilder, E. F.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 2004, 1053, 101–106. (31) Hutchinson, J. P.; Zakaria, P.; Bowie, A. R.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2005, 77, 407–416. (32) Zakaria, P.; Hutchinson, J. P.; Avdalovic, N.; Liu, Y.; Haddad, P. R. Anal. Chem. 2005, 77, 417–423. (33) Hutchinson, J. P.; Macka, M.; Avdalovic, N.; Haddad, P. R. J. Chromatogr., A 2006, 1106, 43–51. (34) Hutchinson, J. P.; Hilder, E. F.; Macka, M.; Avdalovic, N.; Haddad, P. R. J. Chromatogr., A 2006, 1109, 10–18. (35) Hutchinson, J. P.; Hilder, E. F.; Shellie, R. A.; Smith, J. A.; Haddad, P. R. Analyst 2006, 131, 215–221. (36) Connolly, D.; Twamley, B.; Paull, B. Chem. Commun. 2010, 46, 2109– 2111. (37) Xu, Y.; Cao, Q.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2010, 82, 3352– 3358.

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ago,38,39 their use for functionalization of monoliths is rather new. For example, Hilder et al. adsorbed positively charged polystyrene latex nanoparticles on the monolith containing negatively charged sulfonic acid functionalities via electrostatic interaction.30 Haddad’s group has used this approach for the preparation of columns for ion chromatography and capillary electrochromatography (CEC).31-35,40-42 The well-known affinity of gold toward the amino and thiol functionalities was recently used for attachment of gold nanoparticles on the monolithic column. For example, Conolly et al. immobilized gold nanoparticles on the modified poly(butyl methacrylate-co-ethylene dimethacrylate) monolith with photografted vinyl azlactone and modified with either cysteamine or ethylenediamine.36 We have modified poly(glycidyl methacrylateco-ethylene dimethacrylate) monolith with cysteamine and immobilized gold nanoparticles to prepare a new type of stationary phase for the selective isolation of cysteine containing peptides37 and to obtain monoliths with exchangeable chemistries.43 The reduction of sample complexity using selective enrichment methods plays a significant role in proteomics, especially in the characterization of analytes present in very low concentrations, such as phosphoproteins and phosphopeptides.44-47 Protein phosphorylation is one of the most important posttranslational modifications as approximately 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. Thus, the identification of phosphorylation sites in proteins has become very important to understand various cellular processes associated with this specific protein modification. Mass spectrometry is typically used to characterize phosphoproteins after their proteolytic digestion. However, carrying out mass spectrometry of phosphopeptides in the presence of nonphosphorylated peptides is difficult due to ionization suppression by the latter, which decreases ionization efficiency of phosphopeptides and results in low signal intensity. Therefore, efficient isolation and enrichment of the phosphorylated peptides prior to MS detection is needed to increase sensitivity and achieve more accurate characterization of protein phosphorylation. The most common strategy for isolation of phosphopeptides is based on the use of immobilized metal ion affinity chromatography (IMAC), employing ionic interactions between phosphomonoester groups on phosphopeptides and divalent or trivalent transition metal ion, such as Ga3+ and Fe3+ complex with a ligand immobilized on the stationary phase.48 However, this strategy can be negatively affected by the leaching of metal ions during use or storage. Therefore, metal oxide affinity chromatography (MOAC) has recently received more attention since the (38) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801– 1809. (39) Stevens, T. S.; Langhorst, M. A. Anal. Chem. 1982, 54, 950–953. (40) Zhang, S.; Macka, M.; Haddad, P. R. Electrophoresis 2006, 27, 1069–1077. (41) Glenn, K. M.; Lucy, C. A.; Haddad, P. R. J. Chromatogr., A 2007, 1155, 8–14. (42) Haddad, P. R.; Hilder, E. F.; Evenhuis, C.; Schaller, D.; Pohl, C.; Flook, K. J. Abstr. Pap. Am. Chem. Soc. 2009, 236. (43) Cao, Q.; Xu, Y.; Liu, F.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2010, DOI: 10.1021/ac1015613. (44) Leitner, A. Trends Anal. Chem. 2010, 29, 177–185. (45) Reinders, J.; Sickmann, A. Proteomics 2005, 5, 4052–4061. (46) Thingholm, T. E.; Jensen, O. N.; Larsen, M. R. Proteomics 2009, 9, 1451– 1468. (47) Han, G.; Ye, M.; Zou, H. Analyst 2008, 133, 1128–1138. (48) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598– 599.

stationary phases are more stable and exhibit better specificity.29,44,49 The selective interaction of phosphopeptides with metal dioxides via bidentate binding at the dioxide surface eliminates the need for a regeneration step with the metal ions after each analysis as is often required for IMAC sorbents. Although, titanium dioxide and zirconium dioxide are the sorbents that are currently the most widely applied for phosphoprotein and phosphopeptide isolation, new materials and enrichment approaches are desirable to provide a complementary means differing in selectivity and affinity. For example, Mamone et al. recently used hydroxyapatite for the selective isolation of phosphopeptides from a protein digest, demonstrating its potential as an alternative material to the classical IMAC and/or MOAC stationary phases.50 The objective of this work was to develop a monolithic column in a capillary format by incorporating hydroxyapatite nanoparticles. These columns would be used for the separation of a model protein mixture, the detection of aggregates of monoclonal antibodies, and the selective enrichment of phosphopeptides from complex protein digests with off-line MALDI-MS detection. EXPERIMENTAL SECTION Materials and Methods. Hydroxyapatite nanoparticles were purchased from Skyspring Nanomaterials (Houston, TX). 2-Hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), 1-dodecanol, cyclohexanol, 2,2′-azobisisobutyronitrile (AIBN), 3-(trimethoxysilyl)propyl methacrylate, urea, iodoacetamide, dithiothreitol, ammonium bicarbonate, mono- and dibasic sodium phosphate, phosphoric acid, L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin from bovine pancreas, cytochrome c from bovine heart, lysozyme from chicken egg, albumin from chicken egg (ovalbumin), protein A from Staphylococcus aureus, R-casein from bovine milk, β-casein from bovine milk, and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal IgG2 was provided by Pfizer (Chesterfield, MO). HEMA and EDMA were purified by passing them through a column containing basic alumina inhibitor remover (Sigma-Aldrich, St. Louis, MO). Polyimide coated fused-silica capillary (100 µm i.d.) was obtained from Polymicro Technologies (Phoenix, AZ). Instrumentation. All chromatographic experiments were carried out using a liquid chromatography system consisting of an Agilent 1200 Series capillary pump, an Agilent A/D converter 35900E (Agilent Technologies, Santa Clara, CA), and a Linear UVIS-205 absorbance detector. A 6-port nanoscale switching valve (model MXP7980-000) from Rheodyne (Rohnert Park, CA) was used for sample injection. The instrumentation and experimental conditions used for surface area measurements, MALDI/MS analyses, and scanning electron microscopy (SEM)/energy dispersive analysis of X-ray (EDAX) analyses are described in the Supporting Information. Preparation of Monolithic Column with Hydroxyapatite Nanoparticles. The vinylized capillary51 was filled with a mixture containing HEMA, EDMA, 1:3 cyclohexanol-dodecanol, and 1% (49) Zhou, H.; Tian, R.; Ye, M.; Xu, S.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Zou, H. Electrophoresis 2007, 28, 2201–2215. (50) Mamone, G.; Picariello, G.; Ferranti, P.; Addeo, F. Proteomics 2010, 10, 380–393. (51) Krenkova, J.; Gargano, A.; Lacher, N. A.; Schneiderheinze, J. M.; Svec, F. J. Chromatogr., A 2009, 1216, 6824–6830.

AIBN (with respect to monomers, all percentages w/w), containing varying quantities of hydroxyapatite nanoparticles. The thermally initiated polymerization was carried out at 60 °C for 20 h. After polymerization, the monolithic columns were washed with acetonitrile and water and equilibrated in the mobile phase. Protein Separation. A protein mixture composed of ovalbumin, myoglobin, lysozyme, and cytochrome c (0.25 mg/mL each) was dissolved in a solution containing 80% 10 mmol/L phosphate buffer, pH 7.0, and 20% acetonitrile. The mixture was separated using a linear concentration gradient of sodium phosphate buffer and detected at 214 nm. The injected volume was 125 nL. Binding Capacity. The protein binding capacity was determined using frontal elution. Monolithic hydroxyapatite columns were equilibrated with a solution containing 80% 10 mmol/L phosphate buffer, pH 7.0, and 20% acetonitrile; then, a lysozyme solution (0.5 mg/mL) was pumped through the column at a flow rate of 1 µL/min, and absorbance was measured at 214 nm. Binding capacity was calculated at 50% of the final absorbance value of the breakthrough curve and expressed in both mg/mL of column volume and mg/g of hydroxyapatite. Monoclonal IgG2-Protein A Complex. Solutions of monoclonal IgG2 (1.18 mg/mL) and protein A (2 mg/mL) were prepared in 8 mmol/L sodium phosphate buffer, pH 7.0, mixed in various ratios, and incubated for 15 min at room temperature. The IgG2 and its complex with protein A were separated using a monolithic column with encapsulated hydroxyapatite nanoparticles in a linear concentration gradient of sodium phosphate buffer and detected at 214 nm. The injected volume was 125 nL. Phosphopeptide Enrichment. Phosphoprotein digests (1 mg/mL), preparation is described in Supporting Information, were loaded on the hydroxyapatite nanoparticle modified column at a flow rate of 0.5 µL/min for 30 min using a syringe pump. The column was washed with 20% acetonitrile for 15 min followed by water for 15 min. The phosphopeptides were eluted using a 250 mmol/L phosphate buffer, pH 7.0. The eluents were desalted using a C18 ZipTip (Millipore, Billerica, MA), following the protocol suggested by the manufacturer with no concentration step and analyzed in off-line mode by MALDI/MS using DHB in 50% acetonitrile with 1% phosphoric acid as the matrix.52 RESULTS AND DISCUSSION Preparation of Monolithic Column Containing Hydroxyapatite Nanoparticles. The SEM and nitrogen adsorption/ desorption analysis confirmed uniformity of the hydroxyapatite rodlike nanoparticles with an average size of 50 × 150 nm (Figure 1) and a surface area of 100 m2/g. The X-ray photoelectron spectroscopy (XPS) analysis indicates a chemical composition with a Ca/P ratio of 1.67, which is in accord with the structural formula of hydroxyapatite (Ca5(PO4)3OH)2. These commercially available hydroxyapatite nanoparticles were simply dispersed in the typical polymerization mixture, pushed in the capillary, and thermally polymerized. Two methods can be used to modify the monolith with hydroxyapatite nanoparticles. The first approach comprises attachment to the pore surface of the monolith using covalent bonds or electrostatic interactions. The other option is entrapment of the nanoparticles in the porous polymer matrix. Although the (52) Kjellstrom, S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109–5117.

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Figure 1. SEM micrograph of hydroxyapatite nanoparticles used for the preparation of monolithic capillary columns (bar length 1 µm).

former approach may afford monoliths with better accessible nanoparticles and higher coverage of pore surface, the presence of both positively charged calcium groups and negatively charged phosphate groups on the surface of hydroxyapatite nanoparticles causes their agglomeration resulting in dispersion containing micrometer sized aggregates that rapidly plug the column. Unfortunately, a complete redispersion of the aggregated nanoparticles was not achieved despite testing a variety of solvents such as water, phosphate buffer, acetonitrile, acetone, ethanol, and tetrahydrofuran and approaches including sonication and vigorous stirring. The large clusters could be removed using filtration through a 0.22 µm filter but the percentage of nanoparticles in the filtrate was very small to make this dispersion useful. Therefore, attention was focused on the latter method: the entrapment in the monolithic matrix. In order to reduce the nonspecific hydrophobic interaction of proteins and peptides, a polymerization mixture containing hydrophilic 2-hydroxyethyl methacrylate and ethylene dimethacrylate monomers was selected to encapsulate hydroxyapatite nanoparticles in the monolithic capillary column. Composition of Polymerization Mixture. An optimized polymerization mixture should afford a monolithic column that has good permeability and separation performance. Porosity of the monolith directly affects the permeability and largely depends on the percentage of monomers, 2-hydroxyethyl methacrylate and ethylene dimethacrylate, in mixture with porogens consisting of 1:3 mixture of cyclohexanol and dodecanol. Thus, a constant amount of hydroxyapatite nanoparticles (40 mg) was admixed in the polymerization mixtures (250 mg) containing 40%, 30%, and 20% monomers (equal weights of 2-hydroxyethyl methacrylate and ethylene dimethacrylate) providing for nanoparticles/monomer weight ratios of 40/100, 40/75, and 40/50, respectively. These columns were evaluated using a separation of a protein mixture containing ovalbumin, myoglobin, lysozyme, and cytochrome c in a gradient of sodium phosphate buffer to define the quality of the columns. Although the columns were composed of hydroxyapatite and hydrophilic 2-hydroxyethyl methacrylate-based monolith, nonspecific adsorption of proteins assigned to hydrophobicity of the stationary phase was observed. Therefore, 20 vol% acetonitrile was added to all mobile phases consisting of sodium phosphate buffers to minimize the undesired protein and peptide 8338

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Figure 2. Separation of proteins using a monolithic column containing hydroxyapatite nanoparticles at a nanoparticles/monomer ratio of (A) 40/100, (B) 40/75, and (C) 40/50. Conditions: monolithic columns, 12 cm × 100 µm i.d.; mobile phase A, 80% 10 mmol/L sodium phosphate buffer pH 7.0 + 20% acetonitrile; mobile phase B, 80% 500 mmol/L sodium phosphate buffer pH 7.0 + 20% acetonitrile; linear mobile phase gradient 0-100% B in A in 5 min; flow rate, 1.5 µL/min; injection volume, 125 nL; protein concentration, 0.25 mg/mL; UV detection, 214 nm. Elution order ovalbumin (1), myoglobin (2), lysozyme (3), cytochrome c (4).

Figure 3. Separation of proteins using monolithic column containing hydroxyapatite nanoparticles at a nanoparticle/monomer ratio of (A) 0/50, (B) 30/50, and (C) 60/50. Conditions: monolithic columns, 15 cm ×100 µm i.d., mobile phase A, 80% 10 mmol/L sodium phosphate buffer pH 7.0 + 20% acetonitrile; mobile phase B, 80% 250 mmol/L sodium phosphate buffer pH 7.0 + 20% acetonitrile. For other conditions, see Figure 2.

interaction within the monolithic columns. Figure 2 shows that the best separation performance was achieved with a column prepared from the mixture containing the lowest percentage of monomers. This is understandable since more hydroxyapatite particles are buried within the polymer at high monomer percentages, making them inaccessible for proteins. On the basis of the previous experiments, further optimization was carried out using the polymerization mixture containing 20% monomers. Varying amounts of hydroxyapatite nanoparticles were added to the mixture, and monolithic columns were prepared with a nanoparticle/monomer ratio of 0/50-60/50. Figure 3 demonstrates that increasing the proportion of hydroxyapatite nanoparticles in a polymerization mixture improves the separations. However, the increase in the amount of hydroxyapatite in the polymerization mixture also leads to a significant increase in flow resistance. (For details, see Figure 1 in Supporting Information.) This fact limits the upper amount of nanoparticles that can practically be encapsulated in the monolithic matrix.

Figure 4. SEM micrographs of monolithic columns containing hydroxyapatite nanoparticles at a nanoparticle/monomer ratio of (A) 0/50, (B) 30/50, and (C) 60/50 (magnification 10 000×) and corresponding energy-dispersive X-ray spectroscopy spectra.

It is worth noting that the separations carried out under optimal conditions and shown in Figures 2C and 3C are achieved in about 6 min. Since the monolithic columns described in this work are unique and, to our best knowledge, no hydroxyapatite stationary phases in comparable HPLC columns have ever been used, we can compare our capillary columns to those packed with much larger hydroxyapatite particles. Separations of similar protein mixtures carried out using columns packed with particulate hydroxyapatite are typically completed in 20-30 min, and the peaks are much broader.53-55 Since these columns are larger, larger sample volumes have to be used as well. SEM/EDAX Analysis. The SEM micrographs of the hydroxyapatite columns shown in Figure 4 clearly illustrate the change in monolith morphology caused by the incorporation of hydroxyapatite nanoparticles. The flow-through pores are smaller, and the size of microglobules with encapsulated nanoparticles is reduced. SEM with EDAX detection allows the estimation of the amounts of encapsulated hydroxyapatite and its exposure at the pore surface. For example, SEM/EDAX analysis of a monolithic column with no hydroxyapatite confirms the presence of only carbon (97.2 atom %) and oxygen (2.8 atom %). In contrast, a monolithic column with a nanoparticle/monomer ratio of 30/50 contains carbon (67.8 atom %), oxygen (17.4 atom %), phosphorus (5.6 atom %), and calcium (9.2 atom %). Even a more significant increase in the presence of oxygen (28.8 atom %), phosphorus (7.8 atom %), and calcium (22.0 atom %) is observed for a monolithic column featuring a nanoparticle/monomer ratio of 50/ 60. Binding Capacity. Breakthrough curves for lysozyme obtained using frontal analysis allow comparison of binding capacities of the columns with those published for commercially available hydroxyapatite sorbents. Figure 5 confirms that the binding capacity increases with an increasing amount of hydroxyapatite nanoparticles in the polymerization mixture. For example, the binding capacity for a column with a nanoparticle/monomer ratio (53) Benmoussa, A.; Lacout, J. L.; Loiseau, P. R.; Mikou, M. Chromatographia 1996, 42, 177–180. (54) Li, F.; Fang, Y. Q. Talanta 2009, 80, 889–894. (55) Cummings, L. J.; Snyder, M. A.; Brisack, K. Methods Enzymol. 2009, 463, 387–404.

Figure 5. Breakthrough curves of lysozyme demonstrating the effect of the amount of hydroxyapatite nanoparticles in the polymerization mixture on binding capacity. Conditions: monolithic columns 15 cm × 100 µm i.d. containing hydroxyapatite nanoparticles at a nanoparticle/monomer ratio: (a) 0/50, (b) 30/50, (c) 40/50, (d) 50/50, and (e) 60/50; mobile phase, 80% 10 mmol/L sodium phosphate buffer pH 7.0 + 20% acetonitrile; flow rate, 1 µL/min; lysozyme solution, 0.5 mg/mL; UV detection, 214 nm.

of 60/50 is 2.9 mg/mL column volume corresponding to 14.6 mg/g hydroxyapatite. This calculation is based on the assumption that all the hydroxyapatite nanoparticles present in the polymerization mixture have been embedded in the monolithic matrix and are available for interaction with the protein. Despite differences in formats, the binding capacity of our monolithic capillary column is comparable with that of commercially available hydroxyapatite sorbents. Porous hydroxyapatite sorbents manufactured by BioRad Laboratories exhibit a binding capacity of 25 mg/g (13.7 mg/ mL) for CHT Type I and 12.5 mg/g (8 mg/mL) for CHT Type II. IBF Biotechnics agarose-hydroxyapatite composite HA Ultrogel has a binding capacity of 7 mg/mL. Clearly, the monitored breakthrough profiles for all monolithic columns differing in the content of hydroxyapatite exhibit a very steep rise. In analogy with our previous result concerning monolithic stationary phases for separation of proteins,51 these Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

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Figure 7. MALDI/MS spectrum of β-casein tryptic digest before (A) and after (B) phosphopeptide enrichment. Peaks with an asterisk represent phosphorylated peptides. Figure 6. Separation of monoclonal IgG2 and its complex with protein A. For conditions, see Figure 3. Chromatograms: (a) 1.18 mg/ mL IgG2, (b) 1.18 mg/mL IgG2 with 0.08 mg/mL protein A, (c) 1.18 mg/mL IgG2 with 0.16 mg/mL protein A, and (d) 1.18 mg/mL IgG2 with 0.32 mg/mL protein A.

steep curves confirm the fast mass transport kinetics typical of monolithic columns. Separation of Monoclonal Antibody Complex. Owing to its simplicity and high selectivity, affinity chromatography using columns with immobilized protein A is the technique most often utilized for the isolation of IgG antibodies. However, this method also poses serious challenges faced during the purification of monoclonal antibodies. Under the elution conditions, antibodies may form aggregates. Also, protein A may leak from the sorbent as a result of the instability of the attachment bond or proteolytic activity of enzymes present in the feed, thus contaminating the purified monoclonal antibodies. The liberated protein A then forms a complex with IgG degrading the quality of the product. A fast screening method for detection of the leakage is, therefore, desirable. A monoclonal IgG2 was spiked with various amounts of protein A, and the mixture was separated using our hydroxyapatite column as shown in Figure 6. A single peak was monitored after injection of nonspiked IgG2. In contrast, an additional peak was observed at a longer retention time after admixing protein A in the IgG2 solution. This peak corresponds to the IgG2-protein A complex and demonstrates that monoclonal antibody and its complex with protein A can be resolved using the monolithic hydroxyapatite column. Upon increasing the amount of protein A added to the IgG2, the peak area of the antibody decreased and almost disappeared at the highest concentration of protein A. Phosphopeptide Enrichment. Tryptic digests of model phosphoproteins including R-casein, β-casein, and ovalbumin were used to demonstrate the potential of the hydroxyapatite capillary columns for the selective isolation and enrichment of phosphopeptides. Phosphorylated proteins and phosphopeptides bind more strongly to hydroxyapatite through calcium ion affinity than their nonphosphorylated counterparts.2 To fish out the phosphopep8340

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tides, the column was loaded with 15 µL of the phosphoprotein digests; then, the retained peptides were released from the column and analyzed using MALDI/MS. β-Casein is the most common model phosphoprotein that contains five phosphorylation sites (phosphoserines), and tryptic digestion is expected to afford two phosphopeptides with molecular masses of 2061.83 Da (1 phosphoserine) and 3122.26 Da (4 phosphoserines), respectively. Figure 7 compares the MALDI spectra obtained from direct analysis of the β-casein digest with that found after selective enrichment using the hydroxyapatite column. Although no phosphopeptides could be detected in the original digest, the mass spectrum of the eluent from the hydroxyapatite column shows good signals for both of the expected phosphopeptides. The third peak in the mass spectrum corresponds to phosphopeptide fragment with a molecular mass of 2556.09 Da containing one phosphoserine and one missed cleavage site in the sequence. The same procedure was also applied to analysis of a tryptic digest of R-casein. Although the two most abundant phosphopeptides were detected in the original digest, Figure 8 confirms that their detection was significantly improved after sample treatment using selective adsorption and preconcentration in the hydroxyapatite column. The resulting mass spectrum includes only peaks corresponding to specific phosphopeptides of R-casein that are summarized in the table in the Supporting Information. To further investigate performance of the monolithic hydroxyapatite column in the selective enrichment, a more complex tryptic digest of ovalbumin, a phosphoprotein that contains two phosphorylated serines at positions 68 and 344 was used. Tryptic cleavage of ovalbumin is supposed to afford two phosphopeptides with masses of 2088.91 and 2901.32 Da, respectively. The MALDI/MS spectrum of the original ovalbumin digest did not reveal the expected phosphopeptides. However, Figure 2 in the Supporting Information demonstrates that both phosphopeptides could be detected after pretreatment using the monolithic column containing hydroxyapatite nanoparticles.

monolithic column with enhanced selectivity for phosphopeptides enables their isolation from mixtures containing a large excess of other peptides. The retained compounds are then released and analyzed. While the binding capacity of the columns is on par with the current state-of-the-art particulate sorbents, the rapid mass transport enables their application for in-line control of biotechnology processes where throughput is critical. In ongoing studies, the use of these new monolithic columns for the separation of a variety of protein complexes is being explored. Studies are also being initiated that will extend the range of nanoparticles and lead to the design of columns with tailored selectivity. ACKNOWLEDGMENT This work was supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences and Engineering Division, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Support of J.K. by Pfizer Inc. is gratefully acknowledged. Figure 8. MALDI/MS spectrum of R-casein tryptic digest before (A) and after (B) phosphopeptide enrichment. Peaks with an asterisk represent phosphorylated peptides.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSIONS The capillary column containing hydroxyapatite nanoparticles embedded in a porous polymer monolith should prove useful in proteomics analysis for the capture of peptides. This new type of

Received for review July 21, 2010. Accepted August 18, 2010. AC1018815

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