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High-Speed, High-Resolution Monolithic Capillary LC-MALDI MS Using an Off-Line Continuous Deposition Interface for Proteomic Analysis Hsuan-shen Chen, Tomas Rejtar, Viktor Andreev, Eugene Moskovets, and Barry L. Karger*
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 341 Mugar, 360 Huntington Avenue, Boston, Massachusetts 02115
High-speed, high-resolution LC separations, using a poly(styrene-divinylbenzene) monolithic column, have been coupled to MALDI MS and MS/MS through an off-line continuous deposition interface. The LC eluent was mixed with r-cyano-4-hydroxycinnamic acid matrix solution and deposited on a MALDI plate that had been precoated with nitrocellulose. Deposition at subatmospheric pressure (80 Torr) formed a 250-µm-wide serpentine trace with uniform width and microcrystalline morphology. The deposited trace was then analyzed in the MS mode using a MALDI-TOF/TOF MS instrument. Continuous deposition allowed interrogation of the separation with a high data sampling rate in the chromatographic dimensions, thus preserving the high resolution of narrow peaks (35-s peak width at half-height) of the fast monolithic LC. No extracolumn band broadening due to the deposition process was observed. Over 2000 components were resolved in a 10-min linear gradient separation of the model sample, and 386 unique peptides were identified in the subsequent MS/MS analysis. The continuous deposition interface allows the coupling of high-resolution separations to MALDI MS without degradation in separation efficiency, thus enabling high-throughput proteome analysis. Several current methodologies for proteome analysis involve high-resolution separations coupled to mass spectrometry. Shotgun proteomics, based on direct analysis of an enzymatic digest of a complex mixture of proteins,1 provides several characteristics superior to conventional 2D gel electrophoresis, including high throughput and the ability to analyze differential expression via isotope labeling.2,3 A complex proteome sample is often fractionated in a first separation dimension, e.g., by cation exchange chromatography or by isoelectric focusing, and then separated by reversed-phase LC, with mass spectrometric detection. To obtain a high dynamic range and to minimize ion suppression in the MS spectra, high separation efficiency is required. * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (617) 373-2867. Fax: (617) 373-2855. (1) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R., 3rd. Anal. Chem. 1997, 69, 767-776. (2) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (3) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376-386. 10.1021/ac048322z CCC: $30.25 Published on Web 03/10/2005
© 2005 American Chemical Society
Reversed-phase LC, using capillary/nanobore columns packed with silica-based stationary-phase particles, is widely used in proteome analysis.4,5 However, shallow and thus long gradients are generally necessary for efficient separation of complex samples, which limits the throughput of the analysis. Detection of lowabundance proteins can also be challenging when analyzing masslimited samples, such as laser-capture microdissected cell samples. To increase the speed, resolution, and sensitivity of the analysis, several new chromatographic approaches have been introduced. In ultra-high-pressure LC, columns are packed with very small C18 particles (∼1.5 µm) and operated at very high pressure (10 000 psi) to reduce analysis time while maintaining high separation efficiency.6,7 A second approach uses monolithic stationary phases instead of microparticles in the separation column.8-10 Capillary columns with silica-based monoliths or poly(styrene-divinylbenzene) (PS-DVB) monolithic stationary phases are commercially available, and both approaches have been shown to provide highspeed and high-resolution separation of complex peptide mixtures. With elevated column temperature, the widths of the chromatographic peaks at half-height are typically only a few seconds. Such sharp peaks allow fast separations with significant improvements in sensitivity. Despite their advantages in separation and analysis, very narrow peaks can be problematic when coupling the LC separation to mass spectrometry. When using electrospray ionization (ESI) in the LC-MS experiments, MS/MS acquisitions are usually triggered by the signal level of precursor ions in survey MS spectra (i.e., data-dependent scanning). The sampling rate of MS survey scanning, determined by the instrumentation and the number of sequential MS/MS acquisitions, is typically a few (4) Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd. Nat. Biotechnol. 2001, 19, 242-247. (5) Wolters, D. A.; Washburn, M. P.; Yates, J. R., 3rd. Anal. Chem. 2001, 73, 5683-5690. (6) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K.; Pasa-Tolic, L.; Veenstra, T. D.; Lipton, M. S.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2001, 73, 1766-1775. (7) Tolley, L.; Jorgenson, J. W.; Moseley, M. A. Anal. Chem. 2001, 73, 29852991. (8) Premstaller, A.; Oberacher, H.; Walcher, W.; Timperio, A. M.; Zolla, L.; Chervet, J. P.; Cavusoglu, N.; van Dorsselaer, A.; Huber, C. G. Anal. Chem. 2001, 73, 2390-2396. (9) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (10) Tanaka, N.; Kimura, H.; Tokuda, D.; Hosoya, K.; Ikegami, T.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Shintani, Y.; Furuno, M.; Cabrera, K. Anal. Chem. 2004, 76, 1273-1281.
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seconds per spectrum. Since the LC-ESI MS analysis is an online process, the spectra might not be acquired at the maximum of the chromatographic peak, resulting in a loss of sensitivity. In separations of complex peptide mixtures, the common problem of coelution of peptides causing some precursor ions to be completely missed11 is even more pronounced with narrow LC peaks such as with the ultra-high-pressure LC or monolithic LC. An alternative approach to coupling LC separations to mass spectrometry is MALDI MS.12,13 Compared to LC-ESI MS, off-line coupling of LC to MALDI MS by depositing LC eluents onto MALDI targets removes the time constraint for the selection of precursor ions in on-line MS/MS acquisition. Since the separation is fixed on the MALDI plate, MS acquisition and data analysis of the complete LC-MS data set can be completed prior to the MS/MS analysis, allowing an optimized selection of precursor ions. Moreover, MS/MS spectra can be acquired at the maximum of the chromatographic peak, and redundant acquisitions can be minimized. Various approaches have been developed for the coupling of LC separations to MALDI MS.12-21 Off-line fraction collection is the most commonly used approach. The LC eluents can be collected into vials or directly deposited onto a MALDI target, and fraction collectors and robotic workstations for deposition are already commercially available.22,23 However, these devices typically deposit spots pooling 5-30 s of LC eluent, which can cause a significant loss in chromatographic resolution for monolithic LC separations with peak widths under 5 s. Recently, several studies reported spotting at higher rate (2-3 s);18,21 however, an even higher MS sampling rate in the chromatographic time domain may be required for the high-resolution/narrow peak widths of chromatograms currently possible in proteome analysis. Previously, we demonstrated successful use of a continuous deposition interface for coupling capillary electrophoresis to MALDI MS, with MS scanning at the rate equivalent to 4 points/s across narrow electrophoretic peaks.24 In the present study, the continuous deposition device has been applied in the off-line coupling of monolithic LC column separations with elevated (11) Liu, H.; Sadygov, R. G.; Yates, J. R., 3rd. Anal. Chem. 2004, 76, 41934201. (12) 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. (13) Parker, K. C.; Patterson, D.; Williamson, B.; Marchese, J.; Graber, A.; He, F.; Jacobson, A.; Juhasz, P.; Martin, S. Mol. Cell. Proteomics 2004, 3, 625659. (14) Walker, K. L.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 4197-4204. (15) Miliotis, T.; Kjellstrom, S.; Nilsson, J.; Laurell, T.; Edholm, L. E.; MarkoVarga, G. J. Mass Spectrom. 2000, 35, 369-377. (16) 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. (17) Zhang, B.; McDonald, C.; Li, L. Anal. Chem. 2004, 76, 992-1001. (18) 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. (19) Preisler, J.; Hu, P.; Rejtar, T.; Moskovets, E.; Karger, B. L. Anal. Chem. 2002, 74, 17-25. (20) Preisler, J.; Hu, P.; Rejtar, T.; Karger, B. L. Anal. Chem. 2000, 72, 47854795. (21) Tegeler, T. J.; Mechref, Y.; Boraas, K.; Reilly, J. P.; Novotny, M. V. Anal. Chem. 2004, 76, 6698-6706. (22) Bodnar, W. M.; Blackburn, R. K.; Krise, J. M.; Moseley, M. A. J. Am. Soc. Mass Spectrom. 2003, 14, 971-979. (23) Montgomery, H.; Francis, S.; Sekiya, S.; Gaskell, S. J.; Tanaka, K. Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 23-27, 2004.
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column temperature (60 °C) to MALDI MS and MS/MS for the analysis of complex proteomic samples. This paper details the modifications to the previous design of the continuous deposition device to optimize deposition of gradient separations. An in-gel digest of a gel band from an SDS-PAGE separation of a yeast lysate was used as a model sample to illustrate the performance of the continuous deposition interface. The results demonstrate that a continuous streak provides a simple approach to acquire MS spectra with a high sampling rate for narrow chromatographic peaks, thus preserving the high efficiency of fast separations using monolithic columns. EXPERIMENTAL SECTION Chemicals. Acetonitrile (ACN), acetone, trifluoroacetic acid (TFA), ammonium citrate, ammonium bicarbonate, dithothreitol (DTT), iodoacetamide, and R-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma (St. Louis, MO). The CHCA matrix was recrystallized before use. Nitrocelluose was obtained from Bio-Rad Laboratories (Hercules, CA). The tryptic digest of cytochrome c was supplied by Michrom Bioresource (Auburn, CA), and the yeast lysate was purchased from Genotech (St. Louis, MO). Sequencing-grade trypsin was obtained from Promega (Madison, WI). Sample Preparation. SDS-PAGE separation was carried out on a NuPAGE electrophoresis system (Invitrogen, Carlsbad, CA). NuPAGE Novex 4-12% Bis-Tris gel and NuPAGE MOPS SDS running buffer were chosen for the separation of yeast lysate proteins. Approximately 40 µg of the yeast lysate was loaded on the gel, followed by a 30-min electrophoretic separation at 200 V. The gel was then stained using SimplyBlue SafeStain (Invitrogen). The gel band for the molecular mass range of roughly 40-50 kDa was excised and then destained by washing the gel sequentially with pure water, pure ACN, and finally 0.1 M ammonium bicarbonate. In-gel digestion was performed following a standard protocol.25 Briefly, the destained gel was incubated with 200 µL of 25 mM ammonium bicarbonate, 10 mM DTT solution and heated at 56 °C for 1 h. Once reduction was complete, the supernatant was removed from the vial. An aliquot of 200 µL of 55 mM iodoacetamide was then added, and the vial was placed in the dark for 30 min at room temperature. After alkylation, the gel was washed sequentially with 200 µL of 50 mM ammonium bicarbonate solution, 50% ACN/water (v/v) solution and then pure ACN. Next, the gel was dried using a CentriVap concentrator (Labconco, Kansas City, MO), followed by addition of 200 µL of 5 ng/µL of trypsin, and then incubated overnight at 37 °C. To extract the peptides from the gel, 100 µL of extraction buffer (1% TFA, 50% ACN/water (v/v)) was added to the vial, followed by sonication for 10 min. The supernatant was collected and stored at -20 °C prior to the LC-MALDI MS analysis. Continuous Deposition Interface. Figure 1 presents the inhouse-built deposition interface, previously developed for coupling capillary electrophoresis to MALDI TOF MS,24 which has been adapted to the higher flow rates of monolithic LC columns. Prior to the deposition, the stainless steel MALDI plate was spin-coated with nitrocellulose by placing 0.8 mL of 1 mg/mL nitrocellulose (24) Rejtar, T.; Hu, P.; Juhasz, P.; Campbell, J. M.; Vestal, M. L.; Preisler, J.; Karger, B. L. J. Proteome Res. 2002, 1, 171-179. (25) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440-1448.
Figure 1. Diagram of the subatmospheric off-line continuous deposition system. The inset expands the orientation of the deposition capillary in contact with the moving plate. Details can be found in the Experimental Section.
in 98.5% acetone/water (v/v) on the plate, followed by spinning the plate (300-400 rpm) in order to remove excess liquid and to form a thin layer of nitrocellulose (thickness less than 5 µm in SEM picture). The precoated MALDI plate was then placed on an X-Y translational stage inside a deposition chamber. During the process, the deposition capillary was fixed in gentle contact with the MALDI target, and the MALDI plate was moved in a serpentine manner, controlled by a LabVIEW program. Compared to the previous design, the deposition probe was held at an angle of ∼60° to the plate (see the inset of Figure 1). This orientation efficiently eliminated the need for probe rotation at the boundaries of the deposition area. An oil pump (DD-20, Precision Scientific, Chicago, IL) was used to maintain the subatmospheric pressure (∼80 Torr) in the deposition chamber. The pressure was controlled by a needle valve and monitored by a Convectron gauge (model 906, Terranova, Mountain View, CA). The same interface was also used for deposition of sample in the form of spots. In this approach, the deposition capillary was oriented perpendicular to the MALDI plate and moved up and down by an additional stepper motor, also controlled by the LabVIEW program, to deposit the LC eluent as discrete spots. Monolithic LC Separation. Reversed-phase LC separation was carried out on an UltiMate system (Dionex, Sunnyvale, CA). The analytical column was a 100 µm i.d. × 5 cm PS-DVB monolithic column (Dionex), maintained at a constant temperature of 60 °C in the column oven of the UltiMate system during the separation. The sample was injected directly into the analytical
column and separated by a 10-min linear gradient at a flow rate of 1 µL/min. Mobile phase A was 0.1% TFA/water (v/v), and mobile phase B was 0.1% TFA, 50% ACN/water (v/v). The MALDI matrix solution (7 mg/mL CHCA, 0.1% TFA in 70% ACN/water (v/v)) was delivered by a syringe pump (model 101, KD Scientific, New Hope, PA) at a flow rate of 1.5 µL/min to a postcolumn microTee (Upchurch, Oak Harbor, WA) to mix with the LC eluents. The total flow rate of sample/matrix mixture was 2.5 µL/ min, with the MALDI plate moving at a speed of 1 mm/s. MS and MS/MS Acquisition. All MS and MS/MS spectra were acquired by an AB 4700 Proteomic Analyzer (Applied Biosystems, Framingham, MA), as previously described.26 Briefly, the coordinates of a series of adjoining rectangular areas (“wells”) along the deposited streak were submitted to the control software (4700 Explorer, Applied Biosystem). The size of a well was 1 mm × 0.25 mm, representing 1 s in chromatographic time at the plate moving speed of 1 mm/s. Each well was irradiated by a UV laser operating at 200 Hz to acquire the MS spectrum, using 50 laser shots at each of 10 random positions within the well, resulting in a total of 500 laser shots over the whole well. Individual MS spectra were read from the Oracle database of the 4700 Analyzer and combined into a single binary file. The binary file was subsequently analyzed by the laboratory-developed algorithms, MEND27 and PRESEL,28 to generate a candidate peak list for MS/MS analysis. The MS/MS spectra, each acquired using 1000 laser (26) Moskovets, E.; Chen, H. S.; Pashkova, A.; Rejtar, T.; Andreev, V.; Karger, B. L. Rapid Commun. Mass Spectrom. 2003, 17, 2177-2187.
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shots, were processed by standard AB software and then submitted to MASCOT database searching software (ver. 1.9, Matrix Science, London, UK) for peptide and protein identification. RESULTS AND DISSCUSSION New chromatographic approaches, such as monolithic LC and ultra-high-pressure HPLC, provide high-speed and high-resolution separations; however, detection of the narrow chromatographic peaks without degradation of chromatographic performance using MALDI MS can be a challenge. A separate study on the modeling of the influence of the sampling rate of chromatographic peaks has shown that a low sampling rate could cause a significant loss in sensitivity of LC-MALDI MS analysis. It was found that, for 2, 5, and 10 data points collected per chromatographic peak, the theoretical loss in sensitivity could be as high as 50%, 10%, and 3%, respectively.28 On the other hand, acquiring more data points requires a prolonged acquisition time for the larger set of MS spectra. Considering the need to minimize MS acquisition time while maintaining high resolution, 5 points/chromatographic peak was found to be a reasonable compromise.28 Several groups have demonstrated methods of coupling LC separations to MALDI MS, in which the LC eluent is deposited as a series of discrete spots.15,18 To increase the chromatographic sampling rate, more spots would have to be deposited in a given chromatographic time period. However, as the spotting rate is increased, the area density of sample in individual spots is reduced, resulting in a loss of sensitivity. Using a prestructured MALDI plate with hydrophilic sample anchors29 can effectively concentrate sample and reduce the spot size, thus preventing the loss of sensitivity; however, the water-insoluble matrix, CHCA, is not fully compatible with prestructured MALDI plate.30 In contrast, the continuous deposition approach deposits the LC eluent as a continuous trace, in which the area density of sample is constant. The equivalent of a higher chromatographic sampling rate can be achieved by acquiring more MS spectra along the trace, without affecting the sensitivity. The maximum sampling rate is determined by the spatial resolution of the MALDI MS instrument, i.e., the laser spot size. Continuous Deposition. In continuous deposition, the morphology of the deposited traces can be greatly affected by many factors, including matrix composition, hydrophilicity of the MALDI plate surface, deposition flow rate, pressure in the deposition chamber, and speed of the plate movement. To achieve a reproducible MALDI MS analysis, optimization of the deposition parameters is necessary. In the previously developed CE-MALDI MS system, the deposited eluent dried rapidly at the low flow rate (
