Microdeposition Device Interfacing Capillary Electrochromatography

Anal. Chem. 2004, 76, 6698-6706

Microdeposition Device Interfacing Capillary Electrochromatography and Microcolumn Liquid Chromatography with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Tony J. Tegeler, Yehia Mechref, Kirk Boraas, James P. Reilly, and Milos V. Novotny*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve. CEC eluent is transported to the matrix reservoir via a capillary that is connected to the deposition capillary by the connection sleeve inside the matrix reservoir. This connection sleeve also acts as a mixing chamber, allowing the CEC eluent to be mixed with matrix prior to deposition. Complex glycan mixtures can be separated by CEC using hydrophilic-phase monolithic columns, with capillary eluent being deposited on a standard MALDI plate along with a suitable matrix solution. Thousands of discrete, highly homogeneous dots can be generated for a subsequent mass spectrometric analysis. With minor modifications, this device is also applicable to capillary LC of peptides using gradient elution. In this configuration, the outlet of the LC column is connected to a deposition capillary inside a matrix reservoir through a connection sleeve that allows mixing of the LC effluent with an appropriate matrix. The device has been evaluated with the tryptic digests of proteins. The current and future investigations of biological processes are likely to demand increased sophistication of both separation methodologies and mass spectrometry (MS). Proteome complexity is particularly evident in mammalian systems where posttranslational modifications of proteins abound, creating numerous protein forms that may originate distantly from a single gene. Protein glycosylation is a significant posttranslational modification of highly organized and sophisticated eukaryotic systems. In contrast with simple protein modifications, the structural analysis of glycoproteins and their detection in complex biological mixtures may demand the best proteomic1-5 and glycomic methodologies.6-8 * Corresponding author. E-mail: [email protected]. Phone: (812) 8554532. Fax: (812) 855-8300. (1) Saraf, A.; Yates, J. R., III. Protein Arrays, Biochips, Proteomics 2003, 233253. (2) Giometti, C. S. Adv. Protein Chem. 2003, 65, 353-369. (3) Issaq, H. J. Adv. Protein Chem. 2003, 65, 249-269.

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Whether the separation of proteins from complex mixtures is accomplished through a time-honored approach of two-dimensional (2-D) gel electrophoresis or one of the recently investigated 2-D solution alternatives (for a review, see ref 9), the separated fractions are typically digested to peptides prior to their MS investigations. For a preselected pool of glycoproteins, a more labor-intensive approach is involved, which includes protease digestion into mixtures of peptides and glycopeptides and a release of complex glycans from the glycopeptides that all must be separated and structurally identified. Both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are widely used in conjunction with MS for analyzing both peptide mixtures and oligosaccharide pools. A miniaturized ESI source has a distinct advantage of procedural simplicity in capillary LC-based combinations with MS. However, this is less true with capillary electromigration techniques in which the separation systems must be electrically insulated.10-13 With all ESI-based investigations, the aliquot is entirely consumed in one LC/MS analysis. Consequently, MS/ MS data can be lost during a rapid on-the-fly selection of precursor ions or due to detector saturation. Depending on the nature and complexity of investigated proteins, this may decrease the number of identified components and their sequence coverage. At present, capillary LC/MS using nanospray ESI is widespread in proteomic laboratories, while capillary electrochromatography (CEC) using a similar type of ESI interface has recently been introduced into the investigation of complex glycan samples.14-16 (4) Naylor, S.; Kumar, R. Adv. Protein Chem. 2003, 65, 217-248. (5) Smith, R. D.; Anderson, G. A.; Lipton, M. S.; Masselon, C.; Pasa-Tolic, L.; Udseth, H.; Belov, M.; Shen, Y.; Veenstra, T. D. Adv. Protein Chem. 2003, 65, 85-131. (6) Reinhold: V. N.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (7) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-451. (8) Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321-370. (9) Wang, H.; Hanash, S. J. Chromatogr., B 2003, 787, 11-18. (10) Rapp, E.; Jakob, A.; Schefer, A. B.; Bayer, E.; Albert, K. Anal. Bioanal. Chem. 2003, 376, 1053-1061. (11) Spikmans, V.; Smith, N. W.; Tucker, M. G.; Horsten, R.; Mazereeuw, M. LC-GC Eur. 2000, 13, 486-488, 490, 492-494. (12) Walhagen, K.; Gaspari, M.; Tjaden, U. R.; Rozing, G. P.; Van der Greef, J. Rapid Commun. Mass Spectrom. 2001, 15, 878-883. (13) Huber, C. G.; Holzl, G. J. Chromatogr. Libr. 2001, 62, 271-316. (14) Que, A. H.; Mechref, Y.; Huang, Y.; Taraszka, J. A.; Clemmer, D. E.; Novotny, M. V. Anal. Chem. 2003, 75, 1684-1690. (15) Que, A. H.; Novotny, M. V. Anal. Chem. 2002, 74, 5184-5191. 10.1021/ac049341b CCC: $27.50

© 2004 American Chemical Society Published on Web 10/16/2004

The use of MALDI-MS without prior separation is very common in peptide mass fingerprinting and is becoming increasingly common in oligosaccharide mapping and in the structural characterization of simple glycan mixtures (when combined with the use of exoglycosidases).17 However, several recent studies using microdeposition devices have enabled a “display” of previously separated components and facilitated a more detailed analysis of protein/peptide mixtures.18-29 Even when the integrity of a microcolumn separation becomes somewhat compromised by the sample deposition procedure, several distinct advantages accrue: (a) decoupling MS/MS data acquisition from chromatographic separation and independent optimization of MS measurements; (b) a repeated analysis of the same sample regions, if required, and storage of the deposited samples for future investigations; (c) increased dynamic range of solute detection; and (d) reduction of undesirable competitive ionization/desorption effects (widely observed in the analysis of complex mixtures). Moreover, as shown recently by Peters et al.,18 a system based on pulsed electric field deposition can be effectively automated and integrated into a high-performance proteomic platform. Decoupling the separation and mass spectrometer components of the overall analysis could increase flexibility in using alternative mass analyzers for biological samples. This report describes a sample deposition procedure and a system developed for investigations of complex oligosaccharide mixtures by MALDI-MS. The glycans released from selected glycoproteins are first separated by a hydrophilic-phase CEC and deposited onto standard MALDI plates together with a suitable matrix as densely spaced sample dots. With the monolithic CEC columns run isocratically under the conditions of an MS-compatible mobile phase, sample/matrix dots as small as 70 µm make it feasible to deposit several chromatographic runs on a single plate. A custom-designed chamber was constructed for mixing the CEC eluent with a matrix solution. An x-y translational platform was constructed to move the MALDI plate target for uniformly spaced, (16) Que, A. H.; Novotny, M. V. Anal. Bioanal. Chem. 2003, 375, 599-608. (17) Mechref, Y.; Novotny, M. V. Anal. Chem. 1998, 70, 455-463. (18) Peters, E. C.; Brock, A.; Horn, D. M.; Phung, Q. T.; Ericson, C.; Salomon, A. R.; Ficarro, S. B.; Brill, L. M. LC-GC Eur. 2002, 15, 423-428. (19) Ericson, C.; Phung, Q. T.; Horn, D. M.; Peters, E. C.; Fitchett, J. R.; Ficarro, S. B.; Salomon, A. R.; Brill, L. M.; Brock, A. Anal. Chem. 2003, 75, 23092315. (20) Johnson, T.; Bergquist, J.; Ekman, R.; Nordhoff, E.; Schu ¨ renberg, M.; Klo ¨ppel, K. D.; Mu ¨ ller, M.; Lehrach, H.; Gobom, J. Anal. Chem. 2001, 73, 1670-1675. (21) Walker, K. L.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 4197-4204. (22) Hsieh, S.; Dreisewerd, K.; Van der Schors, R. C.; Jime´nez, C. R.; Stahl-Zeng, J.; Hillenkamp, F.; Jorgenson, J. W.; Geraerts, W. P. M.; Li, K. W. Anal. Chem. 1998, 70, 1847-1852. (23) Lou, X.; Van Dongen, J. L. J. Mass Spectrom. 2000, 35, 1308-1312. (24) Wall, D. B.; Berger, S. J.; Finch, J. W.; Cohen, S. A.; Richardson, K.; Chapman, R.; Drabble, D.; Brown, J.; Gostick, D. Electrophoresis 2002, 23, 3193-3204. (25) Miliotis, T.; Kjellstro ¨m, S.; O ¨ nnerfjord, P.; Nilsson, J.; Laurell, T.; Edholm, L. E.; Marko-Varga, G. J. Chromatogr., A 2000, 886, 99-110. (26) Lurie, I. S.; Anex, D. S.; Fintschenko, Y.; Choi, W. Y. J. Chromatogr., A 2001, 924, 421-427. (27) Ekstro ¨m, S.; Nilsson, J.; Heldin, G.; Laurell, T.; Marko-Varga, G. Electrophoresis 2001, 22, 3984-3992. (28) Preisler, J.; Hu, P.; Rejtar, T.; Karger, B. L. Anal. Chem. 2000, 72, 47854795. (29) Preisler, J.; Hu, P.; Rejtar, T.; Moskovets, E.; Karger, B. L. Anal. Chem. 2002, 74, 17-25.

contact needle deposition of discrete, small sample dots. This system differs from continuous vacuum deposition on a moving surface,28,29 the piezoactuated microdispenser,25,27,30 as well as the recently described use of a controlled pulsed electric field dispensor.18,19 The effectiveness of our CEC/sample deposition system is demonstrated here through the MALDI-MS analyses of a dextrin ladder and a complex mixture of glycans derived from bile salt-stimulated lipase (BSSL) of human milk. While designing an effective microdeposition device for CEC/ MALDI-MS, investigation of complex mixtures was the primary objective of this work. We also demonstrate here that, following a simple modification of the instrumental setup, the device is also applicable to capillary LC/MALDI-MS. The versatility of this device, when dealing with different concentrations of aqueous and organic solvents, is illustrated with reversed-phase capillary LC runs for the analysis of tryptic peptides derived from ovalbumin and bovine serum albumin (BSA). Finally, the quantitative aspects of the separation/MALDI-MS methods are briefly compared with results obtained using the dried-droplet method where high-mannose N-glycans derived from ribonuclease B and BSA tryptic digest are used as representative samples. EXPERIMENTAL SECTION Materials. Acrylamide and N,N′-methylenebisacrylamide were purchased from Bio-Rad Laboratories (Hercules, CA). Ammonium persulfate, N,N,N′,N′-tetramethylenediamine (TEMED), 3-methacryloxypropyltrimethoxysilane (Bind-Silane), poly(ethylene glycol) (PEG, MW 10000), cation-exchange resin (H+ form), 2-mercaptoethanol, dihydroxybenzoic acid (DHB), R-cyano-4-hydroxycinnamic acid, ribonuclease B, ovalbumin, BSA, and lyophilized trypsin were obtained from Sigma (St. Louis, MO). Vinylsulfonic acid (sodium salt, 25% (v/v)), 2-cyanoethyl acrylate (CEA), formamide, 1% aqueous formic acid, ammonium hydroxide, and borane-ammonia complex were purchased from Aldrich (Milwaukee, WI). C18 cartridges were received from LCPackings (Sunnyvale, CA), and 20-µL SP20SS resin was from Supelco (Bellefonte, PA). Ammonium carbonate and sodium phosphate monobasic were products of J.T. Baker, Inc. (Phillipsburg, NJ). Sodium phosphate, dibasic was purchased from Mallinckrodt (Paris, KY); micropipet tips (1-10 µL), acetonitrile, methanol, and hydrochloric acid were from Fisher Scientific (Fairlawn, NJ). Dextrin DE 10 was received from Fluka (Buchs, Switzerland). N-Glycosidase F (PNGase F), a recombinant enzyme cloned from Flavobacterium meningosepticum and expressed in Escherichia coli, was purchased from Glyko (Novato, CA). A purified sample of BSSL was kindly provided by Dr. Peter Påhlsson (Department of Biomedicine and Surgery, Division of Clinical Chemistry, University Hospital, Linko¨ping, Sweden). Methods. Release of N-Linked Glycans from Ribonuclease B. A 20-µL aliquot of ribonuclease B (1 µg/µL) was combined with 20 µL of phosphate buffer (10 mM, pH 7.0) containing 1% 2-mercaptoethanol. The solution was then heated for 5 min at 95 °C. After allowing the solution to cool to room temperature, a 2-µL aliquot of PNGase F was added. The solution was then incubated at 37 °C for 3-4 h. After incubation, the glycan solution was passed (30) Laurell, T.; Nilsson, J.; Marko-Varga, G. J. Chromatogr., B 2001, 752, 217232.

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through a C18 cartridge (Waters, Milford, MA) to remove the proteins. Subsequently, the solution was evaporated using a Centrivap concentrator, and glycan residues were then redissolved in acetonitrile/water (50/50, v/v) before separation. Release of N- and O-Linked Oligosaccharides from BSSL. N- and O-linked oligosaccharides were released from the glycoprotein using our modified β-elimination procedure.31 Briefly, 100 µg of BSSL was dissolved in 100 µL of borane-ammonia complex solution, prepared by dissolving 5 mg of borane-ammonia complex (Aldrich, Milwaukee, WI) in 1 mL of 28% aqueous ammonia solution. The reaction mixture was then incubated at 45 °C for 18-24 h. Next, the reaction mixture was evaporated to dryness and resuspended in 20 µL of water. The sample was then desalted using a desalting microcolumn, which was prepared using a micropipet tip (1-10 µL, Fisherbrand) packed with a 40-µL volume of the cation-exchange resin (H+ form) on top of 20 µL of SP20SS resin using glass wool as a frit. A 200-µL volume of aqueous eluent was collected and evaporated to dryness. The residual boric acid was removed through successive additions of 200 µL of methanol and evaporation. The glycan sample was finally reconstituted in acetonitrile/water (50/50, v/v) prior to CEC analysis. Tryptic Digestion of Ovalbumin and BSA. Lyophilized trypsin was reconstituted in 1 mM hydrochloric acid to yield a 1 mg/mL final concentration. A 5 µg/µL solution of either ovalbumin or BSA was prepared in 100 mM ammonium carbonate (pH 8.3), and 0.1 µL of the enzyme solution was added prior to incubation at 37 °C for 18 h. The peptide-containing solutions were then diluted in 0.1% aqueous formic acid solution prior to capillary LC analysis. CEC Column Preparation. Columns were prepared according to the previously described procedures.15,32,33 Briefly, fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with 100-µm i.d. and 360-µm o.d. were used to make CEC columns. The stationary phase for the hydrophilic columns consisted of 5% T, 60% C, 3% PEG, 30% CEA, and 10% vinylsulfonic acid. The polymerization mixture was prepared by dissolving 12.0 mg of acrylamide, 30 mg of N,N′-methylenebisacrylamide, 11.7 µL of CEA, 12.4 µL of vinylsulfonic acid, and 30 mg of PEG in 0.5 mL of formamide and 0.5 mL of 100 mM Tris-150 mM boric acid (pH 8.2). Here, % T refers to the total monomer concentration (g/mL × 100) and % C refers to the degree of cross-linking (g/g × 100).34 Polymerization was initiated using 4 µL of 20% (v/v) TEMED and 4 µL of 40% ammonium persulfate, which were added to 0.5 mL of the above monomer solution (heated to 50°C). The polymerization was allowed to proceed overnight at room temperature. Finally, columns were flushed and conditioned using 50/50 (v/v) acetonitrile/5mM phosphate buffer at pH 3.0. Instrumentation. CEC Setup. The instrument for capillary electrochromatography with the MALDI microdeposition device was assembled in-house from commercially available components. Its schematic is illustrated in Figure 1. The instrument utilized a high-voltage power supply (0-40 kV) from Spellman High Voltage (31) Huang, Y.; Mechref, Y.; Novotny, M. V. Anal. Chem. 2001, 73, 6063-6069. (32) Que, A. H.; Konse, T.; Baker, A., G.; Novotny, M. V. Anal. Chem. 2000, 72, 2703-2710. (33) Que, A. H.; Palm, A.; Baker, A. G.; Novotny, M. V. J. Chromatogr., A 2000, 887, 379-391. (34) Hjerte´n, S. Arch. Biochem. Biophys. 1962, (Suppl. 1), 147-151.


Figure 1. (a) Schematic illustration of the CEC/MALDI plate setup: (1) connection sleeve; (2) outlet reservoir; (3) nitrogen line; (4) separation capillary; (5) inlet reservoir; (6) platinum electrodes; (7) MALDI plate; (8) deposition conduit; (9) matrix reservoir; (10) septum. (b) Schematic illustration of the LC/matrix mixing chamber: (1) connection sleeve; (3) nitrogen line; (8) deposition conduit; (9) matrix reservoir. (c) Enlarged view of the connection sleeve: (1) separation capillary (100-µm i.d., 365-µm o.d.); (2) 400-µm opening; (3) 160µm opening; (4) deposition capillary (25-µm i.d., 365-µm o.d.); (5) silica seal tight tubing sleeve (330-µm i.d., 1.6-mm o.d.).

Electronics (Plainview, NJ). Samples were injected electrokinetically at femtomole levels. The inlet and outlet buffer vials, which contained the mobile phase used for the separation, were manufactured in-house, as described below. Platinum wire electrodes were inserted into the buffer vials using finger-tight nuts. A constant pressure ranging from 5 to 30 psi inside the buffer vials was maintained using nitrogen gas. This pressure maintained a continuous flow between the outlet and matrix reservoirs, thus eliminating (minimizing) sample losses during sample transfer between the reservoirs. The separation capillary (100-µm i.d.) and a deposition conduit (25-µm i.d.) were connected using the connection sleeve (described below), which is located inside the outlet and matrix reservoir vials. Eluent from the CEC column was mixed with the MALDI matrix (20 mg/mL DHB prepared in 80/20 methanol/water) in the connection sleeve and deposited,

Figure 2. Crystallization of the microdrops deposited by the device: (a) dot consisting of 20 mg/mL DHB in methanol/water (80/20); (b) dot consisting of 20 mg/mL DHB in methanol at a deposition rate of 1 dot/0.8 s; (c) dot consisting of 20 mg/mL DHB in methanol with a deposition rate of 1 dot/8.3 s; (d) stainless steel MALDI plate showing 2500 dots with the numbers indicating the order of deposition.

at variable rates, as discrete dots on the MALDI plates. For example, if each dot represents a 1.54-s fraction, a complete CEC run of 1 h will result in 2500 dots being deposited on a typical MALDI plate (see Figure 2d). Capillary LC Setup. Nanoscale capillary LC separations were performed using an Agilent 1100 Series nanosystem including two pumps (an isocratic pump and a gradient nanopump), microvacuum degasser, and a microautosampler. A 10-port nanovalve connected to a nanovalve actuator from Upchurch Scientific (Oak Harbor, WA) was employed for sample and buffer distribution. A 5-µL aliquot of each sample, at a 0.1 µg/µL concentration, was injected and preconcentrated on a 0.3 mm i.d. × 5 mm precolumn cartridge (LCPackings, Sunnyvale, CA). Separations were performed on prefritted, 100 µm i.d. × 16 cm columns (New Objectives,Woburn, MA) packed in-house with C18 silica, having a mean particle diameter of 5 µm and a mean pore diameter of 300 Å (Macherey & Nagel, Du¨ren, Germany). The gradient pump was programmed to deliver initially 5-40% acetonitrile over 40 min and then 40-80% over 2 min at a flow rate of 250 nL/min. Mobile phase A consisted of 0.1% formic acid in 3/97 acetonitrile/ water (v/v), while mobile phase B consisted of 0.1% formic acid in acetonitrile. A custom-designed reservoir (Figure 1b) was constructed similarly to the matrix reservoir shown in Figure 1a, with the exception of the inlet and outlet CEC vials. The packed capillary column was connected to an intermediate sleeve prior to interfacing with the connection sleeve inside the pressurized vial. Eluent from the LC column was mixed with the MALDI

matrix (15 mg/mL R-cyano-4-hydroxycinnamic acid prepared in 60/40 acetonitrile/water with 0.1% formic acid) in the connection sleeve and deposited as discrete dots on the MALDI plates. Each dot represented a 2.5-s fraction of a 1-h LC analysis. Accordingly, 1440 dots are deposited in 1 h in a typical run. Connection Sleeve and Junction Setup. A schematic illustration of the connection sleeve is shown in Figure 1c. The separation capillary and delivery conduit were connected using a silica sealtight tubing sleeve (1.6-mm o.d., 330-µm i.d.) in the outlet reservoir. Two sets of openings were made through the connection sleeve using a small drill bit (400-µm o.d.). A 160-µm-o.d. capillary was inserted through one set of openings to act as a gauge ensuring that the delivery conduit and deposition conduit were ∼160 µm apart from each other. This gauge was then removed and the junction was placed inside a pressurized vial, as depicted in Figure 1a. A second silica seal-tight tubing sleeve was then used to connect the delivery conduit with the deposition conduit in the matrix reservoir. A pressurized vial used to contain the buffer was manufactured in-house using Delrin (Auburn Plastics & Rubber, Inc., Indianapolis, IN), a chemically resistant material, with external dimensions of 5.08 cm (height) × 3.81 cm (width) × 3.175 cm (depth) and internal dimensions of 3.175 cm × 0.635 cm × 1.27 cm. A Teflon sheet was used as a gasket to seal the vial and sustain the applied pressure. Finger-tight nuts with proper sleeves were utilized to bring the delivery conduit and the nitrogen gas lines to the pressurized vial. A deposition conduit was attached to the vial via a finger-tight nut. Eluent/buffer mixture was Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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observed to flow from the exit end of the deposition conduit at a rate dependent upon the pressure applied (5-30 psi) to the buffer and matrix reservoirs. This applied pressure allowed the eluent to be deposited on the surface as separate aliquots in the form of small dots (∼100 µm in diameter). Deposition Device. A device for the controlled placement of eluents from the pressure-driven or electrically driven separations was assembled in-house from commercially available components. The device utilizes a cam-driven reciprocating lever arm to position the end of the conduit near or touching the deposition surface. The deposition conduit is attached to one side of a pivoted arm. On the side opposite to the conduit, the lever is in contact with a motor-driven cam. As the latter turns, the deposition conduit periodically approaches the surface, permitting transfer of the eluent to the surface. A computer-controlled translation stage continuously moves the plate as the eluent emerges from the conduit, thus allowing its deposition as a series of small dots. Typical diameters of the dried dots were approximately 100 µm for CEC and 1 mm for capillary LC. However, dot position, spacing, and size are easily manipulated through varying the speed of the translational stage and the motor-driven cam. MALDI/Time-of-Flight Mass Spectrometry. Mass spectra were acquired on a Voyager-DE RP Biospectrometry Workstation instrument (Applied Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm). MALDI spectra were acquired at 22- and 18-kV accelerating voltage in the positive-ion mode, while the low-mass gate was used to discard the ions with m/z values of less than 800. All acquired spectra were baselinecorrected by using the advanced baseline correction in the Data Explorer software and smoothed by applying a 19-point SavitzkyGolay smoothing routine.35 An in-house Visual Basic program was used to convert the data into a matrix and later plotted with Origin (OriginLabs Corp., Northampton, MA) as three-dimensional chromatograms. The instrument was externally calibrated with the standard dextrin DE 10 ladder and the 4700 proteomics analyzer mass standard kit (Applied Biosystems) for glycan and peptide analyses, respectively. RESULTS AND DISCUSSION Crystallization of Sample Dots. It has been repeatedly shown that sample preparation and deposition play a critical role in MALDI-MS analyses. A “dried-droplet” preparation, employed for water-soluble MALDI matrixes (e.g., 2,5-dihydroxybenzoic acid) on conventional MALDI sample plates, typically results in samples with an irregular crystalline rim (roughly 1-2 mm wide) and a microcrystalline area in the center36 (see Figure 2a). Acceptable mass spectra can only be obtained by irradiating the rim and a subsequent time-consuming search for “sweet spots”. Accordingly, different sample spotting methods have been employed to increase reproducibility and sensitivity of the MALDI analyses, including spot recrystallization with ethanol,37 use of additives forming a more homogeneous spot,38 slow crystallization,39 crushing of (35) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627-1639. (36) Mechref, Y.; Novotny, M. V. J. Am. Soc. Mass Spectrom. 1998, 9, 12931302. (37) Harvey, D. J.; Rudd, P. M.; Bateman, R. H.; Bordoli, R. S.; Howes, K.; Hoyes, J. B.; Vickers, R. G. Org. Mass Spectrom. 1994, 29, 753-765. (38) Karas, M.; Ehring, H.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F.; Grehl, M.; Krebs, B. Org. Mass Spectrom. 1993, 28, 1476-1481. (39) Xiang, F.; Beavis, R. C. Org. Mass Spectrom. 1993, 28, 1424-1429.

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matrix crystals,40 fast evaporation,41 and evaporation under a stream of nitrogen.42 An approach that is more relevant to the small-volume deposition, as expected for CEC and capillary LC analyses described in this study, involves a previously described picoliter and nanoliter dot deposition.43,44 For example, Jespersen et al.44 were able to detect 2.5 amol of bradykinin and cytochrome c by spotting a 250-pL sample aliquot. This low-level detection was mainly attributed to the small volume spotted and the small area of the dried dot. The deposition device described in this study has the ability to deposit samples at low-nanoliter to high-microliter levels, which subsequently results in very small dots. This capability is shown in Figure 2b, where 20 mg/mL DHB MALDI matrix was deposited at a rate of 1 dot/0.8 s and a flow rate of 450 nL/min. Such deposition results in 6 nL of a solvent being deposited as a 100-µm-diameter dot. In addition, the described device offers the capability of varying the deposition volume and, accordingly, the dot size. Increasing the deposition rate to 1 dot/8.3 s resulted in increasing the dot diameter to 400 µm (Figure 2c). Small-dot deposition is highly advantageous, since it eliminates the need for “sweet spot” searching. The typical diameter of a laser in a commercial MALDI mass spectrometer is 25-100 µm, so that when using a 100-µm laser, the entire MALDI dot can be irradiated with the laser during an MS acquisition. Using the best crystallization conditions, 2500 dots were deposited in a zigzag pattern on a flat stainless steel plate, as shown in Figure 2d. Ion Suppression in MALDI/TOF-MS. It is known that the sensitivity of MALDI-MS can be reduced by ion suppression and detector saturation when dealing with complex mixtures.19,28,45 Using N-glycans derived from ribonuclease B as a model system, the importance of chromatographic separation before the MALDI analysis could be demonstrated. Ribonuclease B is a glycoproteincontaining glycan with a common mannose core, (GlcNac)2(Man)x (x ) 5-9). Accordingly, five major m/z values representing these structures are expected to appear in the MALDI-MS spectrum. The importance of separation in reducing ion suppression effects is illustrated in Figure 3, where the mass spectra of a ribonuclease B glycan mixture (Figure 3a) are compared with the mass spectral intensities obtained after CEC separation (Figure 3b). Here, samples prepared via the “dried-droplet” method, produced an m/z value at ∼1909 corresponding to Man9. This was detected with an intensity of 540 counts and a signal-to-noise (S/N) ratio of 16.1 (when the total N-glycan mixture was simultaneously analyzed). The intensity for the same m/z value was increased 6-fold, while the S/N ratio was 2× higher for the CEC-separated mixture (Figure 3b). Accordingly, a CEC separation prior to MALDI analysis significantly improved the detection of minor structures as a result of minimizing, if not eliminating, the ion suppression effect observed with the unfractionated mixture. Any separation technique prior to MALDI-MS analysis is thus likely (40) Xiang, F.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 199-204. (41) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (42) Chan, T. W. D.; Colburn, A. W.; Derrick, P.; Gardiner, D. J.; Bowden, M. Org. Mass Spectrom. 1992, 27, 188-194. (43) Allmaier, G. Rapid Commun. Mass Spectrom. 1997, 11, 1567-1569. (44) Jespersen, S.; Niessen, W. M. A.; Tjaden, U. R.; Van der Greef, J.; Litborn, E.; Lindberg, U.; Roeraade, J. Rapid Commun. Mass Spectrom. 1994, 8, 581-584. (45) Rejtar, T.; Hu, P.; Juhasz, P.; Campbell, J. M.; Vestal, M.; Preisler, J.; Karger, B. L. J. Proteome Res. 2002, 1, 171-179.

Table 1. Reproducibility of Retention tR (min)

Figure 3. (a) MALDI-MS spectra of N-glycans derived from ribonuclease B, using (a) dried-droplet method and (b) CEC separation and deposition of the same glycan mixture using the described device.

Figure 4. 3-D electrochromatogram of Dextrin 10 analyzed by CEC/ MALDI-MS. Experimental conditions: cyano capillary column, 28 cm; mobile phase, 2.4 mM ammonium formate buffer in a 65/35 mixture of acetonitrile/water; field strength, 535 V/cm; injection, 10 kV, 10 s; sample, Dextrin 10 (2.0 µg/µL); matrix, 20 mg/mL DHB in 80/20 methanol/water.

to improve sensitivity and minimize suppression and detector saturation. Reproducibility of CEC/MALDI-MS Analyses. The reproducibility of the deposition device was evaluated using a dextrin ladder. A 3-D recording of the dextrin oligomers separated by a hydrophilic CEC column is shown in Figure 4. The CEC eluent was mixed with a matrix, deposited on a MALDI plate, and analyzed by MALDI/TOF-MS. Due to the capability of these hydrophilic columns to resolve sugar anomers (R and β forms of

solute

run 1

Glc 6 Glc 7 Glc 8 Glc 9 Glc 10

8.73 7.80 8.29 9.24 9.91

Glc 6 Glc 7 Glc 8

7.15 7.55 7.91

run 2

run 3

Run-to-Run (n ) 3) 8.80 8.96 7.96 8.03 8.42 8.57 9.57 9.50 10.11 10.33 Setup-to-Setup (n ) 3) 7.04 7.45 7.36 7.75 7.68 8.30 3.9

RSD 1.3 1.5 1.7 1.9 2.1 2.9 2.6

the sugar on the reducing end),16 dextrin had to be first converted to its corresponding alditols in order to eliminate peak splitting. This conversion was achieved by utilizing a method for the release of N- and O-linked oligosaccharides from BSSL,46 as described in the Experimental Section. Table 1 lists retention times and relative standard deviations (RSD) for the run-to-run and setup-to-setup analyses. The dextrin sample was injected three consecutive times, and retention times (tR) were evaluated for Glc6-Glc10. The setupto-setup evaluation was performed on runs acquired after a complete disassembly and reassembly of the separation capillary and deposition conduit. Disassembly and reassembly were performed three times and the retention times for Glc6-Glc8 determined. The run-to-run RSD values were similar to the previously reported results utilizing hydrophilic monolithic columns and an ESI ion trap MS instrument.15 The setup-to-setup RSD values were slightly higher but are still within the analytically acceptable range. Moreover, the component resolution and peak capacities were not influenced adversely by the described setup, as was concluded from the values attained here, which were similar to what we have previously reported for the same CEC columns using ESI/MS.15,16 CEC/MALDI/TOF-MS Analysis of N- and O-Linked Glycans Derived from Bile Salt-Stimulated Lipase. BSSL is an extremely complex glycoprotein enzyme present in human milk with a wide substrate specificity. The enzyme consists of 722 amino acids, with one site for N-glycosylation at Asn-187. Moreover, there are at least nine O-glycosylation sites near the C-terminus.47 Therefore, the glycan structures of this enzyme cannot be easily evaluated by MS alone due to the heterogeneity of the glycan structures and the possible presence of many structural isomers. This high heterogeneity of the glycan structures is evident in Figure 5, which depicts 3-D and 2-D recordings obtained from the CEC/MALDI/TOF-MS analysis of enzymederived N- and O-glycans. Approximately 50 distinct peaks were displayed in this figure. There are several BSSL peaks that differ by a few mass units, while some other peaks are structural isomers that have been characterized previously in our laboratory.16 Capillary LC/MALDI/TOF-MS Analysis of Protein Tryptic Digests. Ovalbumin and BSA tryptic digest samples were chosen to demonstrate the coupling of LC and MALDI-MS. Both proteins were separately digested with trypsin and separated through (46) Huang, Y.; Konse, T.; Mechref, Y.; Novotny, M. V. Rapid Commun. Mass Spectrom. 2002, 16, 1199-1204. (47) Mechref, Y.; Chen, P.; Novotny, M. V. Glycobiology 1999, 9, 227-234.


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Figure 6. 3-D chromatogram of ovalbumin tryptic digest. For conditions, see Experimental Section.

Figure 5. 3-D electrochromatogram of the mixture of N-linked and O-linked glycans derived from BSSL. Experimental conditions: cyano capillary column, 28 cm; mobile phase, 2.4 mM ammonium formate buffer in a 60/40 mixture of acetonitrile/water; other conditions as in Figure 4.

reversed-phase capillary LC. The microcolumn eluents were mixed with matrix solution and deposited on a stainless steel MALDI plate using the described deposition device. The device typically facilitated the deposition of a dot every 2.5 s. Similarly to the CEC analysis, component resolution and peak capacities for the capillary LC columns used in this study were very comparable to those obtained using the same columns interfaced to ESI/MS (data not shown). Accordingly, it could be concluded that sample transfer in the described setup was highly efficient. The 3-D and 2-D plots of the chromatograms obtained from the LC/MALDI-TOF-MS analysis of the ovalbumin tryptic digest are shown in Figure 6, and Table 2 compares the results obtained with MALDI-MS and LC/MALDI-TOF-MS. The LC/MALDITOF-MS analysis allowed detection of 48 peaks, 16 of which were identified as peptides through the MS-Fit search (SwissProt, http://us.expasy.org/) corresponding to 46% sequence coverage. On the other hand, MALDI-MS produced 24 ions, 9 of which corresponded to peptides. This corresponds to 36% sequence coverage. Those unidentified ions most likely correspond to modified peptides or system peaks that originate in any MS data. Eight peptides were common in both MALDI-MS and LC/MALDI-MS analyses, while one and eight peptides were unique for MALDI/MS and LC/MALDI-MS analyses, respectively 6704 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

(Figure 8). The peptides identified for ovalbumin when analyzed by MALDI-MS and LC/MALDI-MS are in boldface type in the amino acid sequence of ovalbumin (see bottom of Table 2). A similar analysis was performed with the BSA tryptic digest (Figure 7). When the MALDI-MS data were compared with the LC-MALDI-MS using the MS-Fit search, similar results were obtained (Table 2). Here, the LC/MALDI-TOF-MS analysis detected 84 ions, 35 of which were identified, corresponding to 61% sequence coverage. However, the MALDI-MS permitted detection of just 30 ions, 13 of which were identified, accounting to 31% sequence coverage. Twenty-seven peptides were uniquely identified by LC/MALDI-MS, while only three peptides were uniquely identified by MALDI-MS (Figure 8). On the other hand, 10 peptides were commonly identified by both analyses. The peptides identified for BSA, when analyzed by MALDI-MS and LC/MALDI-MS are in boldface type in the amino acid sequence of BSA (see bottom of Table 2). The data clearly demonstrate the advantage of combining separation techniques with MS, where significantly higher sequence coverages are observed. The greater number of peptides identified with capillary LC/MALDI-TOF-MS can be primarily attributed to reduced ion suppression in the MALDI process facilitated by separation and by the fact that isobaric masses cannot be resolved by MS without prior separation of peptides. These effects are particularly pronounced for the analyses of low-abundance peptides. CONCLUSIONS A new type of microdeposition device for MALDI-MS demonstrated in this work facilitates effective sample transfer from multiple CEC runs onto a standard MALDI plate. With minor

Table 2. Comparison of the Sequence Coverage and Peptides Identified for Ovalbumin and BSA Using LC/MALDI-MS and MALDI-MS

modifications to flow rate and matrix solution composition, the device is also compatible with capillary liquid chromatography. The microdeposition device is also well-suited for multiple-plate automation where several runs could be performed on several different samples. Optimization of the mobile phase, flow rates,

deposition volumes, matrix solvents, and plate movement produces dots that are highly homogeneous in size and shape. A minor sacrifice in chromatographic resolution (compared to other means of detection) notwithstanding, the microdeposition device coupled with MALDI-MS typically offers more quantitative Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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Figure 8. Comparison of the number of unique and common peptides identified using MALDI-MS and LC/MALDI-MS for ovalbumin and BSA.

Figure 7. 3-D chromatogram of BSA tryptic digest. For conditions, see Experimental Section.

results and better sequence coverages when compared with a direct MS analysis. Repeated analyses of the same sample dots are feasible, and while not demonstrated in this work, the device should be applicable to any MS system that uses a solid target for a MALDI-MS analysis. Consequently, the same deposited sample could even be sequentially interrogated by MS instruments based on different mass analyzers and featuring different MS resolution and sensitivity. In the analysis of complex peptide mixtures, the use of MALDI is likely to find an increasing application in conjunction with tandem time-of-flight instruments (e.g., TOF/TOF). In a highthroughput fashion, the structural analysis of major mixture components could perhaps be accomplished without a prior (48) Alley, W. R.; Tegeler, T. J.; Mechref, Y.; Novotny, M. V., manuscript in preparation. (49) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 48954903.


chromatographic separation. However, as shown in our forthcoming communication,48 the use of two-dimensional capillary LC together with the MALDI deposition device and a TOF/TOF instrument can enhance significantly the analytical information available for highly complex protein digests. In the analysis of complex glycan mixtures, the use of MALDI-MS has been somewhat limited by the lack of separation capabilities.8 We have recently shown,49 with model glycans, the potential of a complete structural analysis of oligosaccharides without the use of exoglycosidase enzymes. However, due to a frequent glycan isomerism and complexity of oligosaccharide mixtures, separation appears mandatory prior to the use of TOF/TOF-MS. The conjunction of CEC separation with sample microdeposition as demonstrated in this work thus seems potentially beneficial. ACKNOWLEDGMENT This work was supported by Inproteo, Inc., Indianapolis, IN, formerly Indiana Proteomics Consortium, a collaborative effort of Indiana University, Eli Lilly Company, and Purdue University. We thank Dr. Jan Muzikar for his extensive computer work on the visualization of spectra. Received for review May 4, 2004. Accepted August 20, 2004. AC049341B

Microdeposition Device Interfacing Capillary Electrochromatography

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