Combining Liquid Chromatography with MALDI Mass Spectrometry

emerges as a viable technique for routine on-line LC/MALDI ... techniques including blotting the effluents out of a capillary tube ... 4, February 15,...
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Anal. Chem. 2004, 76, 992-1001

Combining Liquid Chromatography with MALDI Mass Spectrometry Using a Heated Droplet Interface Boyan Zhang,† Chris McDonald, and Liang Li*

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 µL/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 µL/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/ MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrowbore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 106 for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) have been widely used for generating biomolecular ions for mass spectrometric analysis. For proteomics applications, these two methods are complementary to each other. * To whom correspondence should be addressed. E-mail: Liang.Li@ ualberta.ca. † Current address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.

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Compared to ESI, MALDI offers higher tolerance toward sample contaminants such as buffers, salts, and surfactants, higher speed of analysis, and lower sample consumption for each analysis. However, MALDI is not readily amendable to combine with solution-based separation techniques, as is the case for ESI. Earlier attempts of using continuous-flow probe1-7 and aerosol interface8-10 have demonstrated that it is possible to combine liquid chromatography (LC) with on-line MALDI detection, but the overall analytical performance in terms of resolution and mass measurement accuracy or detection sensitivity is not sufficient for proteomics applications. Other on-line interfacing methods including moving wheel11 and moving ball12 have also been reported, with varying degrees of success. So far, there seems no on-line interface emerges as a viable technique for routine on-line LC/MALDI experiments. On the other hand, several techniques have been reported to collect the effluents from LC or capillary electrophoresis (CE), followed by off-line MALDI analysis.10 The simplest way of carrying out LC or CE/off-line MALDI is to fractionate and collect the effluents in an array of microtubes/vials or a 96-well microtiter plate. The fractions are then concentrated by methods such as SpeedVac, and the concentrated individual fractions are spotted onto the MALDI plate with matrix for subsequent MALDI MS analysis. However, this method is time-consuming and not readily automated. In addition, there is a risk of sample loss during the concentration and sample/matrix preparation steps. Direct deposition of LC or CE effluents either as discrete spots or as a continuous track on a MALDI plate has been reported.13-30 Several techniques including blotting the effluents out of a capillary tube (1) Li, L.; Wang, A. P. L.; Coulson, L. D. Anal. Chem. 1993, 65, 493-495. (2) Nagra, D. S.; Li, L. J. Chromatogr., A 1995, 711, 235-245. (3) Whittal, R. M.; Russon, L. M.; Li, L. J. Chromatogr., A 1998, 794, 367-375. (4) He, L.; Liang, L.; Lubman, D. M. Anal. Chem. 1995, 67, 4127-4132. (5) Chang, S. Y.; Yeung, E. S. Anal. Chem. 1997, 69, 2251-2257. (6) Murray, K. K.; Caldwell, K. L.; Sheffer, J. D.; Lawson, S. J. The 46th ASMS Conference on Mass Spectrometry and Allied Topics, 1998; p 798. (7) Zhan, Q.; Gusev, A.; Hercules, D. M. Rapid Commun. Mass Spectrom. 1999, 13, 2278-2283. (8) Murray, K. K.; Russel, D. H. Anal. Chem. 1993, 65, 2534-2537. (9) Fei, X.; Wei, G.; Murray, K. K. Anal. Chem. 1996, 68, 1143-1147. (10) Murray, K. K. Mass Spectrom. Rev. 1997, 16, 283-299. (11) Preisler, J.; Foret, F.; Karger, B. L. Anal. Chem. 1998, 70, 5278-5287. (12) Preisler, J.; Hu, P.; Rejtar, T.; Karger, B. L. Anal. Chem. 2000, 72, 47854795. (13) Preisler, J.; Hu, P.; Rejtar, T.; Moskovets, E.; Karger, B. L. Anal. Chem. 2002, 74, 17-25. (14) Orsnes, H.; Graf, T.; Degn, H.; Murray, K. K. Anal. Chem. 2000, 72, 251254. 10.1021/ac034934s CCC: $27.50

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to a plate (e.g., Probot microfraction collector, LC Packings, Amsterdam, The Netherlands), the use of piezoelectric flowthrough microdispenser,27,28 vacuum-assisted deposition,11-13 and electric field-driven droplet deposition29 have been developed. This direct deposition approach reduces sample loss and simplifies the process of sample/matrix preparation. It is particularly compatible with nanoflow separation techniques such as capillary LC or CE and is very useful in dealing with a low quantity of sample. To handle relatively high flow LC effluents, Wall and co-workers30 have recently reported the use of a heated nebulizer31-33 to produce a fine spray of droplets that rapidly evaporate onto a MALDI plate to form a sample track. They have shown that this interface can handle a flow rate of 200 nL/min to 50 µL/min, resulting in a 1-1.5-mm-wide sample track on a MALDI plate precoated with a matrix layer.30 However, in the nebulizer interface, analyte loss can occur due to the difficulty of collecting all the effluent in the form of fine mist exiting the nozzle onto MALDI plate, particularly in high-flow experiments. We report herein a novel interface to combine microbore or narrow-bore column LC separation with MALDI-TOF and MALDI Qq-TOF for peptide and protein analysis. The analytical performance of the technique is assessed, and the application of this technique in proteome analysis of cell extracts is demonstrated. EXPERIMENTAL SECTION Materials and Reagents. Bacterial cells, Escherichia coli (ATCC47076), used in this work were cultured in-house. Bacterial sample was incubated in LB (BBL, Becton Dickinson) at 30 °C with shaking. The E. coli cells were harvested at 48 h, washed with sterile water, lyophilized, and stored below 0 °C before extraction. R-Cyano-4-hydroxycinnamic acid was obtained from Sigma-Aldrich Canada (Markham, ON, Canada) and recrystallized (15) Keough, T.; Takigiku, R.; Lacey, M. P.; Purdon, M. P. Anal. Chem. 1992, 64, 1594-1600. (16) Castoro, J. A.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. J. Am. Chem. Soc. 1992, 114, 7571-7572. (17) Walker, K. L.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 4197-4204. (18) van Veelen, P. A.; Tjaden, U. R.; van der Greef, J.; Ingendoh, A.; Hillenkamp, F. J. Chromatogr. 1993, 647, 367-374. (19) Weinmann, W.; Parker, C. E.; Detarding, L. J.; Papac, D. I.; Hoyes, J.; Przybylski, M.; Tomer, K. B. J. Chromatogr., A 1994, 680, 353-361. (20) Zhang, H. Y.; Caprioli, R. M. J. Mass Spectrom. 1996, 31, 1039-1046. (21) Zhang, H. Y.; Stoeckli, M.; Andren, P. E.; Caprioli, R. M. J. Mass Spectrom. 1999, 34, 377-383. (22) Eckerskorn, C.; Strupat, K.; Kellermanna, J.; Lottspeich, F.; Hillenkamp, F. J. Protein Chem. 1997, 16, 349-362. (23) Kassis, C. E.; DeSimone, J. M.; Linton, R. W.; Remsen, E. E.; Lange, G. W.; Friedman, R. M. Rapid Commun. Mass Spectrom. 1997, 11, 1134-1138. (24) McLeod, G. S.; Axelsson, J.; Self, R.; Derrick, P. J. Rapid Commun. Mass Spectrom. 1997, 11, 214-218. (25) Griffin, T. J.; Gygi, S. P.; Rist, B.; Aebersold, R.; Loboda, A.; Jilkine, A.; Ens, W.; Standing, K. G. Anal. Chem. 2001, 73, 978-986. (26) Lou, X.; van Dongen, J. L. J. Mass Spectrom. 2000, 35, 1308-1312. (27) Miliotis, T.; Kjellstro¨m, S.; Nilsson, J.; Laurell, T.; Edholm, L.-E.; MarkoVarga, G. J. Mass. Spectrom. 2000, 35, 369-377. (28) Miliotis, T.; Ericsson, P.-O.; Marko-Varga, G.; Svensson, R.; Nilsson, J.; Laurell, T.; Bischoff, R. J. Chromatogr., B 2001, 752, 323-334. (29) 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. (30) 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. (31) Biemann, K.; Gagel, J. J. U.S. Patent 4843243, 1989. (32) Biemann, K. U.S. Patent 5770272, 1998. (33) Prevost, T. H.; Dwyer, J. L. U.S. Patent 5772964, 1998.

from ethanol (95%) at 50 °C before use. HPLC grade acetonitrile (ACN) and methanol were from Fisher Scientific (Fair Lawn, NJ). Analytical grade trifluoroacetic acid (TFA) and formic acid were purchased from Caledon Laboratories (Edmonton, AB, Canada). Ammonium bicarbonate was from Sigma (St. Louis, MO). Dithiothreitol was bought from Bio-Rad. Iodoacetamide (Sigma ultrapure) was from Sigma-Aldrich Canada. Water used in all experiments was obtained from a Milli-Q Plus purification system (Millipore, Bedford, MA). All of the peptides and proteins used in this work were purchased from Sigma-Aldrich Canada. E. coli Protein Extraction and Digestion. Bacterial extracts were prepared by a solvent suspension method.34 The lyophilized bacterial cells (6 mg) were suspended in 2 mL of 10 mM TrisHCl buffer (pH 7.85) in a Falco tube and sonicated for 1 min with a Probe tip sonicator (Branson Sonifier 450, Branson Ultrasonics, Danbury, CT) while the Falco tube was immersed in an ice bath. The suspension was centrifuged at 11750g for 10 min. The supernatant (i.e., the clear solution above the cell debris) was then transferred into a fresh vial. This extraction process was repeated 3 times per sample, and the extracts were pooled to maximize the extraction efficiency. The pooled extracts were filtered using Microcon-3 filters with 3000-Da molecular mass cutoff (Millipore) and then concentrated to 1.0 mL by Speed-Vac. Protein content was quantified with a Bio-Rad protein kit based on the Bradford method using bovine plasma γ-globulin as a protein standard. Dithiothreitol was added to the protein mixture to 1 mM final concentration and then incubated at 37 °C for 0.5 h. The proteins were acetamidated by adding 2 mM (final concentration) iodoacetamide and incubated absent from light for 0.5 h. Finally, a small amount of freshly prepared trypsin solution (trypsin content equal to 1/100th of the total protein in the mixture) was added to the protein mixture, incubated at 37 °C for overnight, and then stopped by acidifying the solution. The resultant peptide mixture was kept in a -78 °C freezer until ready for use. HPLC. Solvent delivery and analyte separations were done on an Agilent (Palo Alto, CA) 1100 series capillary-HPLC equipped with an autosampler. Chromatographic separations were performed with several reversed-phase C8, C18 columns of different sizes (0.3-, 1.0-, and 2.1-mm inner diameter and 150-mm length; column medium of synthetic silica; particle size of 5 µm; pore size of 300 Å), bought from Grace Vydac (Hesperia, CA) and Agilent. The UV diode-array detector was set at 214 nm with 8-nm bandwidth. Gradient elution was performed with solvents A (0.1%, v/v, aqueous TFA) and B (0.1%, v/v, TFA in acentonitrile). The gradient profiles for the standard peptide mixture, protein mixture, and bacteria extracts were optimized to perform the best separation within a certain elution time range prior to analysis and are stated in the text, respectively. LC/MALDI Interface. Figure 1 shows the schematic of the heated droplet interface. It includes a transfer tube with its inlet receiving the LC effluent. Droplets are formed at the outlet of the transfer tube. The transfer tube was constructed by placing a silica capillary (outer diameter 180 µm, inner diameter 50 µm) having a polyimide coating (Polymicro Technologies) inside of a stainless steel tube, with dimensions of 1.6-mm outer diameter, 250-µm inner diameter, and 12-cm length. The capillary extended (34) Wang, Z.; Dunlop, K. Y.; Long, S. R.; Li, L. Anal. Chem. 2002, 74, 31743182.

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Figure 1. Schematic of the heated droplet interface for combining LC with MALDI. TC, thermocouple.

beyond the outlet of the stainless steel tubing for a distance of 7 mm. To supply heat to the stainless steel tubing, a heating wire coil (0.75-mm diameter) was wrapped around the tube over a distance of 2.6 cm, having a total of 20 coils around the stainless steel tube. This stainless steel tube was mounted vertically onto the robotic arm of a movable x-y-z stage (model SF-2120, Advantec MFS, Inc., Dublin, CA), as shown in Figure 1, with a spacing of 4 mm between the outlet of the transfer tube and the MALDI plate. Two types of MALDI plates were used in this work. One MALDI target plate used for low flow rate (50 µL/min) separations, was a gold-coated steel plate with 10 × 10 wells (2.4-mm diameter) (Applied Biosystems, Boston, MA). Both the transfer tube and the MALDI plate were heated in a controlled manner in order to evaporate the carrier solvent without spraying, ionizing, nebulizing, splattering, or uncontrolled boiling of the carrier solvent, all of which can cause an undesired spreading or loss of the sample analyte on the MALDI plate. The MALDI plate was heated by mounting it horizontally on a heating block (Boekel Scientific model 11001, Rose Scientific Ltd., Edmonton, AB, Canada), equipped with an adjustable temperature controller. The plate was heated to evaporate the carrier solvent as the droplets land on the plate. A thermocouple temperature sensor was included on a side of the collection device to monitor its temperature (see Figure 1). 994

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The contents of the transfer tube were heated with the heating coil. The coil was connected to an adjustable temperature controller (Barnant model 689-0010, Barrington, IL). The temperature of the transfer tube was monitored with a thermocouple temperature sensor by placing it midway in the heating coil. To further assist in the evaporation of the carrier solvent, a gas-drying device may be used. As shown in Figure 1, a stream of nonreactive gas such as nitrogen gas was flowed through a drying gas line directed at the outlet of the transfer tube. The gas-drying line was heated with a heating coil wrapped around the line. The operational principle and key settings of the interface are presented and discussed in more detail in the Results and Discussion. MALDI Mass Spectrometry. MALDI MS was performed on a Bruker Reflex III MALDI time-of-flight mass spectrometer (Bremen/Leipzig, Germany) equipped with a SCOUT 384 multiprobe inlet and a 337-nm nitrogen laser operated with a 3-ns pulse in positive ion mode. The sample spot was scanned with the laser beam under video observation, and spectra were acquired by averaging 200-300 individual laser shots and processed with the Bruker supporting software. The data were then reprocessed using the Igor Pro software package for presentation (WaveMetrics, Lake Oswego, OR). MALDI Tandem Mass Spectrometry (MS/MS). Selected peptides were fragmented on an Applied Biosystems/MDS-Sciex QSTAR Pulsar QqTOF instrument equipped with an orthogonal

MALDI source employing a 337-nm nitrogen laser (Concord, ON, Canada). The instrument was operated in positive ion mode, and collision-induced dissociation of peptides was achieved with argon as collision gas. Spectra were acquired and processed using Sciex supporting software and reprocessed with Igor Pro software. MS/MS Spectra Interpretation and Database Searching. Known contaminant peak masses from keratin or matrix clusters were eliminated for each spectrum. Only sample peaks were considered for database searching. Both peptide mapping and the peptide sequencing results were searched for protein identification using the Mascot search program (http:// www.matrixscience.com) and the UCSF Protein Prospector Database (http://prospector.ucsf.edu). Safety Consideration. To capture the evaporating solvent from the hanging droplets and the MALDI target plate, the interface should be operated in a well-vented bench area such as inside a fume hood. RESULTS AND DISCUSSION Interface Operation. The heated droplet interface involves the formation of hanging droplets at the outlet of a heated transfer tube. The temperature of the transfer tube is controlled to evaporate as much of the carrier solvent as possible from the droplets without spraying. The droplet dislodges and lands on the heated MALDI plate one by one. The temperature of the MALDI plate is kept above the boiling point of the least volatile solvent, such as water in a solvent mixture of water and acetonitrile commonly used for LC separation of peptides and proteins. As soon as a droplet lands on the plate, the remaining solvent in the droplet evaporate rapidly, depositing the analyte in a small area on the MALDI plate. Since the analyte is transferred from the interface to the plate via droplets and all droplets are collected on the plate, there is no loss of sample during the transfer process. The temperature of the flowing liquid through the transfer tube is carefully controlled (see Figure 2 for schematic illustrations). This temperature can be optimized according to several parameters, including the solvent composition of the flowing liquid, as well as its flow rate. If the temperature of the flowing liquid is not sufficient, there will be little to no evaporation of the solvent as it hangs from the outlet, and large droplets will form (Figure 2A). This will increase the spot size on the MALDI target, reducing the spatial concentration of the analyte. The temperature of the flowing liquid is adjusted just high enough to perform significant evaporation (Figure 2B). This value is usually just below the solvent’s boiling point, to prevent the formation of a large number of gas vapor bubbles inside the transfer line, which can cause the formation of a spray (Figure 2C). As the heated liquid exits the transfer tube, it forms a hanging droplet that clings to the end of the transfer tube outlet by virtue of surface tension. The overall rate of growth of the droplet is controlled by two differential parameters: (1) the rate at which material is added to the droplet and (2) the rate at which material is evaporated from the droplet. The rate at which material adds to the droplet is determined by the volume flow rate of the liquid that is continuously flowing from the transfer tube. Under optimal conditions, and depending on the volume flow rate, solvent evaporation from the hanging droplet can eliminate up to 90% of the total solvent flowing into the droplet (see below). The hanging droplet thus acts as a near-wall-less

Figure 2. (A) Schematic of the transfer tube delivering droplets that are too large for optimal solvent evaporation from the hanging droplet, at a condition where the temperature of the flowing liquid stream in the transfer tube is too low. (B) Schematic of the transfer tube delivering continuously replaced, hanging droplets of the liquid stream that result when the temperature of the flowing liquid stream is appropriately adjusted. (C) Schematic of the transfer tube delivering a conical spray of droplets, as opposed to continuously replaced hanging droplets, resulting when the temperature of the flowing liquid stream significantly exceeds the boiling point of the carrier solvent. Table 1. Some Representative Experimental Parameters for the Heated Droplet Interface at a LC Flow Rate of 40 µL/min solvent composition 10% ACN/90% water 50% ACN/50% water 90% ACN/10% water

heating effluent exit droplet coil droplet flow rate dropping temp (°C) temp (°C) (µL/min) rate (Hz) 270 245 240

52 50 46

12 8 4

0.5 0.3 0.2

container for concentration of analytes. Since the rate of material going into the droplet is higher than the rate of solvent evaporation, the droplet will grow in volume until a critical size is reached, at which point gravity overcomes the surface tension holding the droplet. At this point, the bulk of the hanging droplet falls off the end of the transfer tube and is collected on the MALDI plate directly below the outlet (Figure 2B shows the optimum condition). In practice, the extent of heat applied to the transfer tube is adjusted according to the solvent composition of the flow stream and flow rate. An example of temperature settings for the transfer tube is listed in Table 1. This experiment was performed at a fixed flow rate of 40 µL/min, and there was no nitrogen drying gas directed on the hanging droplets. The carrier solvent composition was varied from 10 to 90% acetonitrile, and the optimum temperature of the heating coil was explored. The heating coil temperature was measured by a thermocouple sensor inserted at the midpoint of the heating coil. The droplet temperature was measured by a thermocouple placed directly under the exit of Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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the capillary, in direct contact with the falling drops. The effluent flow rate was calculated by collecting the solvent in a vial for 10 min. During a HPLC experiment with varying compositions of carrier solvent over a predetermined duration of separation (i.e., gradient separation), the temperature of the heating coil can be programmed to follow the changes of the solvent compositions in order to maximize solvent evaporation from the hanging droplets without the formation of a spray of fine mist. The heated droplet interface shown in Figure 1 incorporates a block heater to impart heat to the MALDI plate. The temperature of the plate is controlled with the goal of rapidly evaporating the fallen droplets to form a small analyte spot without causing boiling and splattering. The plate was initially heated to 120 °C for starting solvent conditions in a typical HPLC separation of peptides and proteins (2% acetonitrile) and then was gradually adjusted down to 110 °C as the percentage of acentonitrile increased to 90%. The boiling and splattering points of different percentages of water/ acetonitrile solvent mixtures on a MALDI target were investigated. It was found that droplets of pure water would boil and splatter on the heated MALDI plate at plate temperatures above 125 °C. A 50% acetonitrile/water droplet of the same volume boiled at ∼115 °C, whereas a droplet of pure acetonitrile boiled on the plate at 90 °C. These boiling and splattering points are dependent on the droplet size, as well as the starting temperature of the droplet. By heating the LC effluent in the transfer tube, the effluent exited the transfer tube at an elevated temperature. In addition, smaller droplets (about 50-500 nL) fell on the heated MALDI plate, compared to the case where no or little heat is applied to the transfer tube. The combination of these two factors allows for more efficient solvent elimination without solvent splattering and results in the collection of smaller sample spots. During the spotting process, a stream of hot N2 gas (100 °C) can be directed at the outlet end of the transfer tube and the MALDI target, which can further reduce droplet size and aid in solvent elimination. For example, with the LC flow rate of 40 µL/ min at a solvent composition of 50% H2O and 50% acetonitrile, the effluent flow rate after the interface was found to be ∼6 µL/min at the droplet rate of 0.3 Hz, instead of the 8 µL/min found in the case where no hot N2 gas was applied. In general, under the conditions used for peptide and protein separation, the LC effluent could be confined to the 0.8-mmdiameter wells of the custom-built MALDI plate at flow rates of 5-50 µL/min, with 1-min collection time/well. At flow rates up to 200 µL/min, a 1-min fraction could be confined to the 2.4-mmdiameter wells of the commercial MALDI plate from Applied Biosystems. After sample collection is completed, matrix is added to each sample spot for subsequent MALDI experiments. In all experiments, HCCA was used and prepared by using a saturated solution in 50% acetonitrile/49.5% water/0.5% TFA. Other matrixes can also be used in the mass spectrometric analysis, depending on the applications. For the MALDI plate with spots of 0.8-mm diameter, 0.2 µL of matrix was spotted, while 0.5 µL was spotted upon the standard 2.4-mm-diameter spots. It should be noted that a tee connector can be added to the transfer tube to supply a stream of matrix solution, which can then mix with the effluent in the interface. Although this on-line matrix addition approach eliminates the need of matrix deposition after LC sample deposition, 996

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it provides very little flexibility in matrix solvent selection for optimal analyte/matrix cocrystallization. Since the heated droplet interface involves heating of the analyte, one potential concern is thermal decomposition of the analyte. However, for protein and peptide analysis, we have not encountered this problem in hundreds of experiments we have run on the LC/MALDI instrument. This is not surprising considering that boiling proteins in SDS solution is a standard technique for protein denaturing in protein chemistry. The temperatures of the surfaces with which the analytes make contact in the LC interface are generally below 120 °C. In a simple test where a peptide (bradykinin) was deposited to the heated MALDI plate, MALDI spectra collected from the sample spots before and after heating for 60 min were very similar with no visible degradation of the molecular ion signals and no appearance of any additional peaks. Detection Sensitivity. The detection sensitivity of the HPLCMALDI interface is demonstrated from the analysis of a dilute peptide mixture. The standard peptide mixture contained a 1 µM concentration of each of the following: bradykinin, substance P, and adrenocorticotropic hormone (ACTH) 18-39 was diluted 1000 times with MilliQ water to a dilute peptide mixture containing a 1 nM (1 fmol/µL) concentration of each peptide. Five microliters of the diluted peptide mixture was subjected to RP-HPLC separation on a 1-mm-i.d. C8 RP-HPLC column at a flow rate of 40 µL/ min. The effluent was monitored by the UV detector (214 nm) of the Agilent 1100 HPLC system. The effluent after the UV detector was then introduced to the transfer tube and collected on the custom-built stainless steel target (0.8-mm wells) in 1-min fractions. Figure 3A displays the UV chromatogram between 18 and 25 min following the injection of 1 µL of the concentrated peptide mixture (1 pmol of each peptide injected), and Figure 3B is the corresponding UV chromatogram at the same time interval when 5 µL of the dilute mixture was injected (5 fmol of each peptide). The MALDI-MS spectra obtained from the analysis of the fractions collected at these time intervals are shown in Figure 3C. Note that the MALDI spectrum corresponding to 18 min refers to the effluent collected between 18 and 19 min. As well, there is a time delay of up to 40 s between the UV peak appearance and the elution of the peak to the MALDI plate. It can be seen from Figure 3A that a strong UV peak can be detected for each of the three peptides at 1-pmol injection at the time intervals of 18-19, 2223, and 23-24 min. However, the UV signals decreased below the detection limits when 5 fmol was injected (Figure 3B). The corresponding MALDI-MS spectra (Figures 3C) of fractions between 18 and 25 min display strong signals for all three peptides, even at the 5-fmol level. This level of detection closely corresponds to the detection level that is observed when these peptides are individually subjected to direct MALDI analysis (i.e., without HPLC separation) using a conventional microliter sample deposition method. This example thus illustrates that this heated droplet interface does not cause sample loss or the loss of analyte signals. It should be noted that the time interval used for fraction collection can be varied from a few seconds to minutes. A time interval of 1 min is generally used for peptide and protein applications. For peptide separation using microbore or narrowbore columns, the peak width at the baseline is usually about 40 s to 1 min, and for protein separation, the peak is even broader.

Figure 3. LC/MALDI analysis of a mixture of three peptides (bradykinin, substance P, ACTH 18-39). (A) UV trace of the separation, from 18 to 25 min, following injection of the mixture containing 1 pmol of each peptide. (B) UV trace of the separation following injection of the mixture containing 5 fmol of each peptide. (C) Corresponding MALDI spectra from fractions collected over the same time interval and conditions as (B). Where applicable, the peptide peaks are expanded to show the isotopic resolution.

The use of a longer time interval will reduce the chromatographic resolution. On the other hand, the use of a shorter time interval may overly divide an individual peptide component into several fractions. Since a high concentration of analyte in the solid matrix gives a better signal-to-noise ratio in MALDI, dividing a sample into several fractions is not desirable for high-sensitivity detection. In Figure 3C, at the 19-min fraction, the use of a 1-min interval enables all of the bradykinin to be eluted from the peak collected and analyzed by MALDI. Substance P was detected in the 22-min fraction. The extraneous peaks in this spectrum are from the oxidized substance P, as well as some matrix clusters. The worst

case in peak sampling using a 1-min interval is to divide a 4060-s peptide peak in half. As an example, ACTH 18-39 was detected mainly in the 23-min fraction, but a strong analyte signal was also observed in the 24-min fraction (see Figure 3C). In this case, the ACTH chromatographic peak was fractionated to the 23- and 24-min fractions. Another conclusion to be drawn from this experiment is that sample carryover between fractions is nonexistent. For example, there was no bradykinin peak in either the 18- or 20-min fractions and likewise for substance P in the 21or 23-min fractions. Dynamic Range. Detection of a minor peptide component in the presence of a high concentration of other components can be a challenging task. But in many proteomics applications, protein concentrations can vary in the order of 106 or higher. Detection of low-abundance components in a mixture containing highabundance components is required to provide a comprehensive analysis of a proteome. MALDI-TOF is capable of detecting peptides and proteins from low femtomoles to high nanomoles, if they are analyzed individually. But if a mixture of high- and lowconcentration components is analyzed, the low-concentration components may not be detected even if we use an extreme experimental condition where the peaks from the high-concentration components are greatly saturated in the spectrum. The failure of detecting the low-concentration components in this case is not due to the instrumental dynamic range (e.g., digitizer); it is attributed to the ion suppression effect. Ion suppression is a result of several contributing factors such as reduced analyte incorporation in matrix cocrystals for the low-concentration components, preferential desorption of the high-abundance component at the onset of the desorption event, and less favorable competition for ionization in the gas plume for the low-abundance components. This effect was demonstrated in Figure 4, which shows direct MALDI MS analysis of bradykinin (the minor component) in the presence of increasing amounts of ACTH 18-39. In the initial experiments (Figure 4A-C), the amount of bradykinin was kept constant (2 fmol) and the amount of ACTH 18-39 was varied (202000 fmol). As shown in Figure 4A, bradykinin (m/z 1060) was detected by direct MALDI MS in a 10-fold mole excess of ACTH 18-39 (m/z 2465) (the minor peaks at m/z above the bradykinin peak were from matrix/salt clusters). The bradykinin peak was still clearly seen at 100-fold excess of ACTH 18-39 (Figure 4B). However, at a 1000-fold excess of ACTH 18-39, the signal for bradykinin was totally suppressed (Figure 4C). Even at higher loadings of 20 or 100 fmol of bradykinin (Figure 4D and E), which is much higher than the instrument detection limit of this peptide, the presence of 1000-fold excess of ACTH 18-39 continuously suppressed the signal of bradykinin. This simple peptide mixture was then subjected to HPLC separation, followed by sample collection using the heated droplet LC-MALDI interface. Note that there was no analyte detected in the UV chromatogram during the washing step after the separation experiment, suggesting that near 100% analyte was collected. The peptide mixtures were prepared at various ratios (1:100-1:106) of bradykinin/ACTH 18-39 and separated on RP C18 columns of different dimensions (0.3 × 150, 1.0 × 150, and 2.1 × 150 mm). A representative chromatogram is shown in Figure 5A. Bradykinin eluted at 19 min, and ACTH 18-39 eluted in a window from 23 to 26 min. On the 1.0 × 150 mm (microbore) C18 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 4. Direct MALDI analysis of bradykinin in the presence of an excess amount of ACTH 18-39: (A) 1:10 mole ratio of bradykinin to ACTH, with 2 fmol of bradykinin and 20 fmol of ACTH deposited on the target; (B) 1:100 mole ratio, with 2 fmol of bradykinin and 200 fmol of ACTH deposited; (C) 1:1000 mole ratio, with 2 fmol of bradykinin and 2 pmol of ACTH spotted; (D) 1:1000 mole ratio of bradykinin to ACTH, with 20 fmol of bradykinin and 20 pmol of ACTH; and (E) 1:1000 mole ratio, with 100 fmol of bradykinin and 100 pmol of ACTH.

column, as shown in Figure 5B-D, bradykinin could be detected in the presence of 105 excess of ACTH 18-39 (Figure 5C). In this case, a total of 5 µg of peptide sample was injected on the columsthis amount corresponds to the manufacturer’s specified capacity for the column. The amount of ACTH 18-39 injected (5 µg or 2 nmol) was fixed, and the amount of bradykinin injected varied dependent on the corresponding mole ratio (e.g., 20 fmol for 1:105). For the analysis of the peptide mixture of 1:106 bradykinin/ACTH, as shown in Figure 5D, no bradykinin signal was observed. Several other peaks at higher m/z’s were observed and were likely from impurities from the sample. These impurities may play a role in suppressing the bradykinin signal at this low level of sample loading. Nevertheless, the ability to fractionate the majority of the suppressing compound (i.e., ACTH 18-39) by HPLC allows for bradykinin to be detected in the presence of 998 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

Figure 5. LC/MALDI analysis of bradykinin in the presence of varying amounts of excess ACTH 18-39, separated by using a 1-mm C18 column: (A) UV trace recorded for the separation of bradykinin and ACTH (1:1000 mole ratio of bradykinin to ACTH, 5 µg total injected); (B) MALDI analysis of the 1:104 mole ratio, 200 fmol of bradykinin injected; (C) MALDI analysis of the 1:105 mole ratio, 20 fmol of bradykinin injected; (D) MALDI analysis of the 1:106 mole ratio of ACTH to bradykinin, with 2 fmol of bradykinin injected; (E) MALDI analysis of 106:1 ratio of ACTH to bradykinin, 20 fmol of bradykinin and 20 nmol of ACTH injected on a 2.1 × 150 mm column.

a significantly higher proportion of interfering components, as compared to direct MALDI analysis. The ability of detecting low-abundance bradykinin in the mixture is dependent upon the amount of sample present for MALDI analysis, which is dependent upon the sample loading on the HPLC column. A larger column allows for higher sample loading and therefore higher detection dynamic range. For example, a capillary column (0.3 × 150 mm) has a capacity of 0.5 µg. When tested, the mixture of bradykinin and ACTH 18-39 revealed a detectable bradykinin signal only up to 1:104. This corresponds to a loading of 20 fmol of bradykinin and 200 pmol of ACTH 18-39. Although a 1:104 ratio is an improvement over the dynamic range of direct MALDI (1:100), this is lower than that achieved in the 1-mm column experiment (i.e., 1:105). We also examined the detection dynamic range for a 2.1 × 150 mm column, whose sample loading capacity is 25 µg, about

4-5 times that of the 1.0-mm column. When dealing with this narrow-bore column, however, the flow rate must also be increased. The operational flow rate of the 2.1-mm column is 200 µL/min, which is within the operational range of the LCMALDI interface. In this experiment, we purposely overloaded the column by injecting 50 µg (20 nmol) of ACTH 18-39 mixed with 20 fmol of bradykinin (i.e., bradykinin:ACTH 18-39 ) 1:106). From the mass spectrum presented in Figure 5E, it is clear that the bradykinin peak is visible, suggesting that we can achieve a dynamic range of 106. However, at this level of sample loading (i.e., doubling the loading capacity of the column), column overloading is already quite apparent, as there is a significant ACTH 18-39 peak in the fraction (see Figure 5E). In fact, the ACTH 18-39 peak was found throughout the chromatographic separation on the 2.1-mm column. Column overloading led to the flowing of some of the major components through the column with improper retention. This work points out that, while one would expect a dynamic range improvement of using 2.1-mm column over that of 1-mm column by about 4-5-fold, overloading a column can achieve somewhat greater improvement. However, there is a limit that sample can be overloaded to a column. Other factors such as chromatographic resolution and ion suppression due to the overflow of a major component must be considered in real-world applications. The above results illustrate that, with the ability of handling high-flow separation using large columns, the heated droplet LCMALDI interface exhibits a broad detection dynamic range. Six orders of magnitude detection range can be achieved. The advantage of this unique feature is analogous to gel electrophoresis where detecting low-abundance proteins is often achieved by using a large-format gel separation, which allows the loading of a larger amount of starting protein materials for proteome display, compared to a minigel separation. As in gel electrophoresis, sample overloading in LC can only result in a modest amount of improvement in detection dynamic range. E. coli Proteome Analysis. The following example illustrates how the high dynamic range afforded by the heated droplet LCMALDI interface allows for improved performance in the identification of individual analytes from a complex mixture of biological origin. Protein extraction and digestion from an E. coli sample were described in the Experimental Section. The resulting tryptic digest was subjected to LC/MALDI analysis under different experimental conditions for data comparison. In one experiment, a 60-µL sample of the peptide mixture (∼0.4 µg/µL) was loaded on the 2.1 × 150 mm C8 column and collected from 1 to 90 min, 1 min/well, onto a 100-well MALDI plate from Applied Biosystems. The collected sample spots were analyzed by MALDI MS, and strong peaks observed in the MS analysis were subjected to MS/MS analysis by using MALDI Qq-TOF to produce fragment ion spectra. The resulting spectra were used to identify the unknown proteins using the MASCOT search program. Figure 6A shows the UV chromatogram of the tryptic peptide separation from the narrowbore column. The gradient condition (i.e., min:B%: 0:2, 5:2, 7:10, 100:55, and 110:80) was optimized to fractionate the peptides over a long time span so that the effluent can be fractionated and collected from 1 to 90 min using the 100well plate. The large peaks from 4 to 9 min in Figure 6A were

Figure 6. 214-nm UV chromatograms for (A) 24 µg of tryptic extracted bacteria peptides injected to a narrow-bore (2.1 × 150 mm) RP C8 column; (B) 1 µg of the same sample injected to a capillary (0.3 × 150 mm) RP C18 column.

from the salts/buffer components and other small molecules present in the sample. Almost all peptides were detected from spots fractionated from 20 to 80 min. Among them, most peptides were detected only once from the fractionated spots. However, some peptides were seen in multiple fractions. Figure 7A shows an example of one LC fraction at 46 min being characterized by MALDI-TOF MS detection. A representative MS/ MS spectrum of a peptide at m/z 1292.75 obtained by using MALDI Qq-TOF from the fraction is shown in Figure 7B. As shown in Figure 7A, more than 20 peptide peaks were detected; but only 14 peptides were able to produce database-searchable MS/MS spectra. Among these MS/MS spectra, eight significant protein matches (indicated with a star in Figure 7A) have been found by the Mascot search engine. These eight matches identify six unique proteins: catalase HP II (m/z 1291.75), FKBP-type peptidyl-prolyl cis-trans isomerase (m/z 1045.59), 50S ribosomal protein L10 (m/z 1687.81), glutamate decarboxylase R (m/z 1519.77 and 2047.95), chaperone protein DNAK (m/z 1233.56 and 1405.59), and outer membrane protein C precursor (m/z 1348.68). Table 2 provides a summary of the results. Of the 598 MS/MS spectra generated from the analysis of this sample, 254 resulted in significant matches to the protein database, and these peptides are from 133 unique proteins of E. coli bacteria source. For comparison, a capillary C18 column (0.3 × 150 mm) was injected with 2.5 µL of the tryptic peptide mixture (∼0.4 µg/µL). The peptides were eluted with a gradient (min:B%): 0:2, 10:2, 12: 15, 100:50, and 110:80. The UV chromatogram from this separation is shown in Figure 6B. Peptide signals were detected from 18 to 75 min. Using the same protocol as above, the collected sample spots were subjected to MS and MS/MS analysis, followed by searching the fragment ion spectra for protein identification. As summarized in Table 2, the results of this experiment yielded Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 7. (A) MALDI-TOF MS spectrum obtained for the fraction at 46 min from the narrow-bore column separation (Figure 6A). (B) MALDIQqTOF MS/MS spectrum for peptide at m/z 1292.75. Table 2. Summary for LC-MALDI-QqTOF MS/MS Analysis of Water-soluble Protein Components of E. coli Cell Extracts (ATCC47076) LC/MALDI interface

Q-TOF MS/MS analysis result

loading amount: 24 µg HPLC column: narrow-bore RP 2.1 × 150 mm C8 MALDI target: 10 × 10 well (i.d., 2.4 mm) plate

598 MS/MS spectra collected 256 peptide hits 133 unique protein identifications

loading amount: 1 µg HPLC column: microbore RP 0.3 × 150 mm C18 MALDI target: 10 × 10 well (i.d. 0.8 mm) plate

167 MS/MS spectra collected 77 peptide hits 45 unique protein identifications

much less peptides that gave significant match to the protein database. In this case, 167 MS/MS spectra were collected and 77 peptide hits were registered from database searching, which resulted in the identification of 45 unique proteins. The above results illustrate that the use of a larger amount of sample that can be injected and separated on a larger column (at a higher optimal flow rate) can result in the detection of more proteins. Because of the capability of analyzing a larger amount of sample using the current LC/MALDI setup, we anticipate that we can work with a much greater amount of starting materials to enrich low-abundance proteins via multiple sequential prefractionation techniques, compared to that can be handled by a capillary column LC experiment. We are working toward the goal of mapping out the entire proteome of a microorganism such as E. coli using the LC/MS approach. The results shown in this work on the water-soluble proteins of the E. coli extract already demonstrate that the fractionated peptides by HPLC are quite complex. The use of extensive sample prefractionation prior to HPLC/MALDI should reduce the complexity of the peptide 1000 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

mixture and result in much more comprehensive detection of the E. coli proteome. CONCLUSIONS A novel HPLC-MALDI interface has been developed for peptide and protein analysis. It is based on the formation of hanging droplets at the outlet of a transfer tube that accepts LC effluent, the use of heat to partially evaporate the solvents from the droplets, deposition of the droplets to a confined area at a given time interval on a heated MALDI plate, and further evaporation of the solvents from the droplets on the plate to form small analyte spots. MALDI analysis of the fractionated analytes can be carried out with the addition of matrix to the analyte spots. For LC separation with flow rates of up to 50 µL/min and fractionation at a 1-min interval, analyte spot size per fraction is typically less than 0.8 mm in diameter. For a higher flow rate of up to ∼200 µL/ min, analyte deposits from each fraction is generally confined to an area of 2.4 mm in diameter. The interface does not introduce analyte loss and the detection sensitivity of LC/MALDI using this

heated droplet interface is in low femtomoles for peptides, similar to that obtained by standard MALDI sample deposition methods. Since high flow rates can be used with this interface, it is compatible with microbore or narrow-bore column separation, wherein a relatively large amount of sample can be injected. This allows a significant increase in detection dynamic range for peptide mixture analysis, i.e., on the order of 106. The advantage of having broad dynamic range for proteome analysis is illustrated in identification of the water-soluble protein components of E. coli cell extracts.

ACKNOWLEDGMENT This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). B.Z. was supported by a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR).

Received for review August 10, 2003. Accepted December 2, 2003. AC034934S

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