Anal. Chem. 2007, 79, 3312-3319
Microsphere Entrapped Emitters for Sample Preconcentration and Electrospray Ionization Mass Spectrometry Terry Koerner, Ruixi Xie, Feng Sheng, and Richard Oleschuk*
Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
In this study a nano-electrospray emitter is constructed by precisely positioning entrapped octadecylsilane (ODS) particles within a photoinitiated polymer at the exit aperture of a capillary. Following poly-merization, the microsphere/polymer hybrid material is able to withstand pressures greater than 4000 psi for 1 cm length of material. Smaller microspheres (3 µm) patterned at the exit aperture of a capillary generated the most sensitive/ stable electrospray from 100 to 1000 nL/min and moderately stable signal under 100 nL/min. Constant infusion of a standard PPG solution from a batch of eleven emitters resulted in a relatively small variance in total ion current (TIC) counts (8%). The entrapped microsphere emitter design yields an emitter that minimizes clogging and eliminates dead volume between the chromatographic bed and the electrospray emitter. The entrapped ODS microspheres can also be used for sample preparation prior to mass spectrometry (MS) analysis. We show the solidphase extraction and preconcentration of 20-700 fmol of a peptide (leucine enkephalin) prior to MS analysis on an emitter with 1 cm of entrapped microspheres. The field of proteomics has grown significantly in recent years because of the high potential for insight into biological systems.1 Unlike the analysis of DNA, where polymerase chain reaction (PCR) provides an efficient process for amplifying minute amounts of DNA, there exists no parallel process for proteins and peptides. Instead researchers are required to analyze samples that are extremely small in size, limited in concentration, and present among large amounts of other proteins, often several orders of magnitude higher in concentration. This, along with the drive to discover clinical biomarkers and drug discovery targets, has been the impetus for the development of several methodologies for protein analysis. When coupled with conventional high-performance liquid chromatography (HPLC) or LC (µL-mL/min) systems, the electrospray ionization process requires a coaxial sheath flow of heated gas to aid in desolvation due to the relatively high flow rates. The large flow rates result in the formation of larger droplets and thus a competition of analytes for the surface charge of the (1) Lambert, J.-P.; Ethier, M.; Smith, J. C.; Figeys, D. Anal. Chem. 2005, 77, 3771-3787.
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droplets resulting in reduced sensitivity for some analytes. With the development of nanospray techniques, decreased sample size and improved sensitivity result from using pulled fused silica capillaries and reduced flow rates (10-200 nL/min).2,3 Lower flow rates produce smaller droplets, with a larger surface area to volume ratio, which have many benefits compared with conventional electrospray (ESI) flow rates.3,4 Although nanospray emitters have enhanced the analysis of protein, peptides, and noncovalent complexes,5 they are still problematic.6,7 Nanospray emitters require some skill to load and are difficult to fabricate reproducibly, necessitating that each emitter be calibrated. Furthermore, these capillaries have a high tendency to clog, resulting from the trapping of fine particulates in the small capillary tip (1-10 µm), and require continuous replacement. A number of different variations on nanospray emitters have been published, and there has been some improvements in the nano-electrospray emitter coating;3-10 however, the main disadvantages associated with the fundamental design are still an issue. Recently Tang et al. demonstrated that by splitting the sample flow into a number of emitters, the ESI efficiency could be enhanced.11 In this study, multiple spray emitters were fabricated by laser etching 2-9 holes (30 µm diameter) in a thin sheet of polycarbonate that was then coupled to the sample flow. The consequence was enhanced ionization efficiency and sensitivity increases, which are likely due to better desolvation and smaller droplets emanating from the multiple sprayers. Although effective, the device still requires larger flow rates (1000-5000 nL/min) compared with nanoelectrospray applications (10-500 nL/min) and would not be generally useful for applications driven by EOF. (2) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLafferty, F. W. Anal. Chem. 1995, 67, 3802-5. (3) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (4) Juraschek, R.; Dulcks, T.; Karas, M. J. Am. Soc. Mass Spectrom. 1999, 10, 300-308. (5) Zampronio, C. G.; Giannakopulos, A. E.; Zeller, M.; Bitziou, E.; Macpherson, J. V.; Derrick, P. J. Anal. Chem. 2004, 76, 5172-5179. (6) Karas, M.; Bahr, U.; Dulcks, T. Fresenius’ J. Anal. Chem. 2000, 366, 669676. (7) Wetterhall, M.; Klett, O.; Markides, K. E.; Nyholm, L.; Bergquist, J. Analyst (Cambridge, U. K.) 2003, 128, 728-733. (8) Maziarz, E. P.; Lorenz, S. A.; White, T. P.; Wood, T. D. J. Am. Soc. Mass Spectrom. 2000, 11, 659-663. (9) White, T. P.; Wood, T. D. Anal. Chem. 2003, 75, 3660-3665. (10) Trapp, O.; Pearce, E. W.; Kimmel, J. R.; Yoon, O. K.; Zuleta, I. A.; Zare, R. N. Electrophoresis 2005, 26, 1358-1365. (11) Tang, K.; Lin, Y.; Matson, D. W.; Kim, T.; Smith, R. D. Anal. Chem. 2001, 73, 1658-1663. 10.1021/ac0619478 CCC: $37.00
© 2007 American Chemical Society Published on Web 03/28/2007
Furthermore, these devices may prove difficult to manufacture on a larger scale. Recently we showed that patterning a mesoporous material at the end of a capillary could produce a similar effect.12 At high flow rates (200 nL min-1 to 1 µL min-1) porous polymer monolith (PPM) patterned capillaries performed in a manner similar to a conventional emitter with a single visible Taylor cone; however, at flow rates below 200 nL/min a mist, presumably from a number of very small Taylor cones emanating from the mesoporous material, was produced. The PPM electrospray emitter is attractive because the polymer formation is facile and the photoinitiated chemistry allows for precise placement. In addition, simply controlling the composition of the porogenic solvent or modification of the monomer can alter the pore size and surface characteristics.13 Although the PPM electrospray emitters worked well, they possess limited surface area unless the pores are very small and they are not easily functionalized.14 Although the surface area and function are not immediately important for spraying purposes, they will pose a problem if the material is to be used for chromatography in conjunction with enhancing the electrospray process. In this paper we show that a hybrid material composed of octadecylsilane (ODS) chromatographic microspheres entrapped within a polymer matrix yields nanospray emitters that function extremely well over a large flow rate range (10-1000 nL/min). Entrapping chromatographic particles within a capillary has been accomplished previously by sintering the ODS particles15 or entrapping the particles in a silicate matrix,16 a sol-gel matrix,17,18 and even a polymer matrix.19 Each of these methods is timeconsuming, and all prove difficult for precisely patterning microspheres within a specific section of a column. Additionally the methods encapsulate the particles by filling the interstitial spaces between the microspheres with material either organic or inorganic that ultimately leads to encapsulation of the microspheres. Encapsulation prevents interactions between the sample and stationary phase, which results in decreased chromatographic efficiency and increased column back-pressure. Ideally one would like the microspheres trapped with the minimal amount of material to confine the microsphere with the remaining surface left for sample interaction. Polymer matrix entrapment is an attractive alternative since a number of different monomers and cross-linkers are commercially available. In addition we have chosen a photoinitiated process to enable facile control of polymerization by simply illuminating for various periods of time and precise patterning of the material within a capillary. The photopatterned material links the chromatographic material at specific microsphere-microsphere and microsphere-capillary contact points, producing a robust cross-linked chromatographic bed capable of (12) Koerner, T.; Turck, K.; Brown, L.; Oleschuk, R. D. Anal. Chem. 2004, 76, 6456-6460. (13) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1998, 70, 2288-2295. (14) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (15) Asiaie, R.; Huang, X.; Farnan, D.; Horvath, C. J. Chromatogr., A 1998, 806, 251-63. (16) Chirica, G.; Remcho, V. T. Electrophoresis 1999, 20, 50-56. (17) Dulay, M. T.; Kulkarni, R. P.; Zare, R. N. Anal. Chem. 1998, 70, 51035107. (18) Tang, Q.; Xin, B.; Lee, M. L. J. Chromatogr., A 1999, 837, 35-50. (19) Chirica, G. S.; Remcho, V. T. Anal. Chem. 2000, 72, 3605-3610.
withstanding >4000 psi of applied pressure. In addition to using the hybrid material as a nanospray emitter, we show that the tip can be used to perform solid-phase extraction and preconcentration of a peptide sample to further aid in detection. Furthermore, the same microsphere entrapping methodology to produce nanospray emitters can be applied to the fabrication of chromatographic beds for capillary electrochromatography, nano-HPLC, and µ-TAS applications. EXPERIMENTAL SECTION Reagents. All solutions for polymerization and mass spectrometry were prepared in >18 MΩ Milli-Q water (Millipore, Bedford, MA). Butyl acrylate monomer was obtained from Aldrich and filtered through freshly activated alumina to remove the inhibitor hydroquinone monomethyl ether. 3-(Trimethoxysilyl)propyl methacrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1,3-butanediol diacrylate (BDDA), benzoin methyl ether (BME), and leucine enkephalin were all obtained from Aldrich and used as received. Glacial acetic acid and HPLC grade acetonitrile were obtained from Fisher Scientific. Ethanol was purchased from Commercial Alcohols Inc. (Brampton, ON, Canada). Octadecylsilane particles (Microsorb 100-3 C18) were purchased from Varian Canada Inc. (Mississauga, ON, Canada). Emitter Fabrication. A porous polymer monolith retaining frit was prepared in a fused silica capillary (75 µm i.d., 363 µm o.d. with a UV-transparent coating, Polymicro Technologies Phoenix, AZ) using a solution described by Ngola et al.20 In the first step, vinyl groups were grafted to the surface of the capillary to provide sites to anchor the polymer to the walls of the capillary. This was accomplished by filling the capillary with a solution of 3-(trimethoxysilyl)propyl methacrylate (20%; all quantities are volume percent unless otherwise stated), glacial acetic acid (30%), and deionized water (50%) and then left to react for 1 h at room temperature. The capillaries were washed with a solution consisting of ethanol (20%), acetonitrile (60%), and 5 mM phosphate buffer, pH 6.8 (20%) and then stored in the same solution. A polymerization mixture consisting of 23% butyl acrylate monomer, 10% BDDA as the cross-linker, 0.2% AMPS, 0.1% 3-(trimethoxysilyl)propyl methacrylate as additional adhesion promoter, 0.2% (g/ mL) BME as initiator, 13.25% ethanol, 40% acetonitrile, and 13.25% 5 mM phosphate buffer, pH 6.8, as porogenic solvent, which was introduced into the capillary with a Harvard Apparatus 11plus syringe pump (Holliston, MA). The capillary was then covered by aluminum foil, leaving 1.5 mm of the UV-transparent capillary exposed to the Spectroline hand-held UV lamp (model ENF-280C at 365 nm; Fisher Scientific, Nepean, Ontario, Canada), Figure 1A. In the next step a slurry of 3 µm of ODS particles in acetonitrile (5 mg/mL) was packed into the column under pressure with the aid of an ultrasonic bath until at least a 2 cm length of microspheres was obtained, Figure 1B. This process was monitored using a Nikon Eclipse ME600 microscope (Tokyo, Japan). Several column volumes of the same polymerization mixture used to form the PPM frit were then introduced into the capillary. The capillary was masked, exposing only the microsphere region of the capillary. The microspheres were then immobilized by exposing this region, at a distance of 2 cm from the UV lamp, for 2 min (20) Ngola, S. M.; Fintschenko, Y.; Choi, W.-Y.; Shepodd, T. J. Anal. Chem. 2001, 73, 849-856.
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Figure 2. Schematic of PPM and microsphere-based mesoporous ESI emitter.
Figure 1. Schematic of the nanospray emitter fabrication and accompanying optical micrographs (100×). (A) Capillary (75 µM i.d. 360 or 150 o.d.) containing a frit made of a porous polymer monolith. (B) Packing with ODS derivatized silica microspheres (3.0 µm). (C) B after ODS particles have been entrapped. (E) Micrograph showing the entire 1 cm length of the entrapped microspheres and the end of the nanospray emitter.
(254 nm), Figure 1C. The capillaries were then flushed in the opposite direction using a syringe pump with a mixture of 80:20 (v/v) acetonitrile/(5 mM tris buffer, pH 8), to remove the unreacted monomer materials and any loose microspheres. The PPM retaining frit was removed by cleaving the capillary with a ceramic cutter, exposing the end of the 1-1.5 cm length of entrapped microspheres, Mass Spectrometry. All experiments were performed on an API 3000 triple quadrupole mass spectrometer (MDS-Sciex, Concord, Canada) fitted with a nano-electrospray source (Proxeon, Odense, Denmark) consisting of an x-y-z stage and two CCD camera kits to aid in the positioning of the capillary. A liquid junction was used to supply the electrospray voltage, which consists of the microsphere entrapped capillary, a zero-deadvolume stainless steel union (Upchurch-Scientific Products, North York, ON, Canada), and a transfer line. Applied ESI voltages were 3200 V for flow rates ranging from 600 to 1000 nL/min and 3000 V for flow rates
