Sample Preparation for MALDI Mass Spectrometry ... - ACS Publications

BD Biosciences, 2 Oak Park, Bedford, Massachusetts 01730, and Dyax Corp., 300 Technology Square,. Cambridge, Massachusetts 02139. A new method for ...
0 downloads 0 Views 306KB Size
Download PDF
Anal. Chem. 2006, 78, 6160-6168

Sample Preparation for MALDI Mass Spectrometry Using an Elastomeric Device Reversibly Sealed on the MALDI Target Xiaoxi Chen,*,† Anthony Murawski,† Guannan Kuang,‡ Daniel J. Sexton,‡ and William Galbraith†

BD Biosciences, 2 Oak Park, Bedford, Massachusetts 01730, and Dyax Corp., 300 Technology Square, Cambridge, Massachusetts 02139

A new method for improving low-concentration sample recovery and reducing sample preparation steps in matrixassisted laser desorption/ionization mass spectrometry (MALDI MS) is presented. In the conventional approach, samples are typically desalted and/or concentrated with various techniques and deposited on the MALDI target as small droplets. In this work, we describe a new approach in which an elastomeric device is reversibly sealed on the MALDI target to form a multi-well plate with the MALDI target as the base of the plate. The new format allows a larger volume (5-200 µL) of samples to be deposited on each spot and a series of sample handling processes, including desalting and concentrating, to be performed directly on the MALDI target. Several advantages have been observed: (i) multiple sample transferring steps are avoided; (ii) recovery of low-concentration peptides during sample preparation is improved using a novel desalting method that utilizes the hydrophobic surface of the elastomeric device; and (iii) sequence coverage of the peptide mass fingerprinting map is improved using a novel method in which proteins are immobilized on the hydrophobic surface of the elastomeric device for in-well trypsin digestion, followed by desalting and concentrating the digestion products in the same well. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become a powerful and widely utilized analytical tool for peptides, proteins, and many other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids).1-6 In MALDI MS, the preparation and deposition of samples onto the sample plate (MALDI target) is critical to the success of the * Author to whom correspondence should be addressed. Tel.: 781-301-3294. Fax: 781-301-3339. E-mail: [email protected]. † BD Biosciences. ‡ Dyax Corporation. (1) Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology; MCC Press: San Diego, CA, 2003. (2) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422, 198-207. (3) Fenselau, C.; Demirev, P. A. Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom. Rev. 2001, 20, 157-171. (4) Gevaert, K.; Vandekerckhove, J. Protein identification methods in proteomics. Electrophoresis 2000, 21, 1145-1154. (5) Harvey, D. J. Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom. Rev. 1999, 18, 349-450.

6160 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

method.7,8 Sample solutions are usually mixed with a solution of energy absorbing matrix, and a small volume of the combined solution is deposited onto predefined target sites of the MALDI target.6-8 Once deposited, the sample droplet is usually allowed to dry in ambient air (by so-called “dried droplet evaporation”), forming matrix crystals that contain analyte molecules (e.g., peptides and proteins) on the target sites. Because the diameter of the target sites are generally small and often densely packed, small (e.g., 0.5-2 µL) droplets of the liquid solutions are deposited onto the plate target sites to achieve proper sample placement and to avoid sample overlap between target sites. Because MALDI targets are generally flat, various techniques have been developed for attracting and maintaining the liquid samples at the plate target sites.9-15 For example, MALDI plates have been formed with a hydrophobic masking (e.g., poly(tetrafluoroethylene)) over a hydrophilic substrate with the target sites being exposed.10-12 These approaches are still limited by the (6) Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 1988, 60, 22992301. (7) Terry, D. E.; Umstot, E.; Desiderio, D. M. Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J. Am. Soc. Mass Spectrom. 2004, 15, 784-794. (8) Pluskal, M. G.; Bogdanova, A.; Lopez, M.; Gutierrez, S.; Pitt, A. M. Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry. Proteomics 2002, 2, 145-150. (9) McComb, M. E.; Oleschuk, R. D.; Chow, A.; Ens, W.; Standing, K. G.; Perreault, H.; Smith, M. Characterization of hemoglobin variants by MALDITOF MS using a polyurethane membrane as the sample support. Anal. Chem. 1998, 70, 5142-5149. (10) Hung, K. C.; Ding, H.; Guo, B. Use of poly(tetrafluoroethylene)s as a sample support for the MALDI-TOF analysis of DNA and proteins. Anal. Chem. 1999, 71, 518-521. (11) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Prestructured MALDI-MS Sample Supports. Anal. Chem. 2000, 72, 3436-3442. (12) Botting, C. H. Improved detection of higher molecular weight proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on poly(tetrafluoroethylene) surfaces. Rapid Commun. Mass Spectrom. 2003, 17, 598-602. (13) Redeby, T.; Roeraade, J.; Emmer, A. Simple fabrication of a structured matrixassisted laser desorption/ionization target coating for increased sensitivity in mass spectrometric analysis of membrane proteins. Rapid Commun. Mass Spectrom. 2004, 18, 1161-1166. (14) Tannu, N. S.; Wu, J.; Rao, V. K.; Gadgil, H. S.; Pabst, M. J.; Gerling, I. C.; Raghow, R. Paraffin-wax-coated plates as matrix-assisted laser desorption/ ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting. Anal. Biochem. 2004, 327, 222-232. (15) Gundry, R. L.; Edward, R.; Kole, T. P.; Sutton, C.; Cotter, R. J. Disposable Hydrophobic Surface on MALDI Targets for Enhancing MS and MS/MS Data of Peptides. Anal. Chem. 2005, 77, 6609-6617. 10.1021/ac060286b CCC: $33.50

© 2006 American Chemical Society Published on Web 08/04/2006

small volume (5-10 µL) of the liquid solutions that can be deposited onto the plate target sites to achieve proper sample placement and to avoid sample overlap between target sites. In a typical MALDI MS experiment, the sample solution is purified and desalted before deposition on the MALDI target.7,8 The purification and desalting is usually achieved with columns, pipet tips or multi-well filter plates being packed with C18 media or other chromatography media.16-20 A common procedure for desalting samples uses hydrophobic media (such as C18 media) to perform capture and release of peptides or proteins. The sample solutions are flowed through the hydrophobic media; it is expected that the peptides and proteins in the solution be retained in the hydrophobic media by binding to the hydrophobic surfaces during this step. The hydrophobic media then can be washed with a washing buffer; the peptides and proteins are expected to be retained in the media during this step. Finally, an elution solution is passed through the hydrophobic media, which dissociates the peptides and proteins from the media; it is expected that the peptides and proteins flow out with the elution buffer in this step. In this approach, a significant portion of the peptides and proteins in the sample solution may flow through the hydrophobic media without binding to the hydrophobic surfaces. As a result, recovery of the peptides and proteins may be poor, particularly when the sample concentration is low. In proteomics research, MALDI MS has been widely used to analyze protein fragments created by protease digestion such as trypsin digestion.7,8,16-20 The goal is to obtain a peptide mass fingerprinting map for the identification of proteins. Typically, trypsin digestion is performed either in gel (after polyacrylamide gel electrophosis of protein samples) or in solution (after liquid chromatography separation of protein samples), followed by desalting and concentrating the trypsin digestion products. The process usually involves multiple sample transferring steps, which may result in significant sample loss and affect the sequence coverage of the peptide mass fingerprinting map. To enable the deposition of a significantly larger volume of samples directly on the MALDI target and allow the trypsin digestion and sample desalting and concentration processes to be directly performed on the MALDI target, we introduce a new method utilizing an elastomeric device (MALDI sample concentrator). The MALDI sample concentrator can be sealed on the MALDI target to create an array of temporary wells above the corresponding array of target sites. Relatively large volumes of samples can be deposited and processed in the wells. A new desalting method is also introduced for the purpose of maximizing (16) Doucette, A.; Craft, D.; Li, L. Protein Concentration and Enzyme Digestion on Microbeads for MALDI-TOF Peptide Mass Mapping of Proteins from Dilute SolutionsAnal. Chem. 2000, 72, 3355-3362. (17) Craft, D.; Doucette, A.; Li, L. Microcolumn Capture and Digestion of Proteins Combined with Mass Spectrometry for Protein Identification. J. Proteome Res. 2002, 1, 537-547. (18) Nissum, M.; Schneider, U.; Kuhfuss, S.; Obermaier, C.; Wildgruber, R.; Posch, A.; Eckerskorn, C. In-Gel Digestion of Proteins Using a Solid-Phase Extraction Microplate. Anal. Chem. 2004, 76, 2040-2045. (19) Baczek, T. Fractionation of peptides in proteomics with the use of pI-based approach and ZipTip pipet tips. J. Pharm. Biomed. Anal. 2004, 34, 851860. (20) Salplachta, J.; Rehulka, P.; Chmelı´k, J. Identification of proteins by combination of size-exclusion chromatography with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry and comparison of some desalting procedures for both intact proteins and their tryptic digestsJ. Mass Spectrom. 2004, 39, 1395-1401.

the recovery of peptides and proteins at low concentration. This method uses the hydrophobic surface of the wells of the MALDI sample concentrator as the desalting media and allows a lowconcentration sample solution to concentrate and dry on the hydrophobic surface. A new in-well trypsin digestion method is also explored in which the trypsin digestion of proteins is performed with the proteins bound to the hydrophobic surface of the wells of the MALDI sample concentrator. It is expected that, by performing trypsin digestion, desalting, and concentrating in the same well, the sample loss is reduced, compared to other methods with multiple sample transferring steps. EXPERIMENTAL SECTION Materials. One-hundred-position MALDI targets were obtained from Applied Biosystems (Foster City, CA). Microplatesize MALDI targets were obtained from Bruker Daltonics (Billerica, MA). C18 resin-packed pipet tips (ZipTip) were obtained from Millipore (Bedford, MA). Acetonitrile, trifluoroacetic acid (TFA), R-cyano-4-hydroxycinnamic acid (R-cyano), sinapinic acid, human angiotensin II, synthetic peptide P14R, human ACTH fragment 18-39, bovine insulin oxidized B chain, bovine insulin, horse heart cytochrome c, and human hemoglobin were obtained from Sigma (St. Louis, MO). Bovine serum albumin (BSA) trypsin digest was obtained from Michrom Bioresources (Auburn, CA). A trypsin digestion kit was obtained from Pierce (Rockford, IL). MALDI Sample Concentrator. MALDI sample concentrators were manufactured at BD Biosciences Discovery Labware (Bedford, MA). The MALDI sample concentrator is composed of poly(dimethylsiloxane) (PDMS), which is an elastomer that has been used extensively in microfluidic channel designs.21-23 The elastomeric nature of PDMS makes it possible to make reversible connections between the concentrator and the MALDI target that do not leak fluid, compared to other polymer materials that are commonly used to make microplates, such as polystyrene or polypropylene. Figure 1 shows the design of the MALDI sample concentrator. The concentrator has an array of wells (Figure 1A) that match with the spot positions on the MALDI target (Figure 1B). Each well is open both at the top and at the bottom. The size of the bottom openings is the same or smaller than the spot size on the MALDI target (usually defined by circle marks on MALDI targets). The bottom surface of the MALDI sample concentrator is made optically flat (which is achieved by manufacturing the concentrator in a mold with an optically flat surface), and, therefore, it is easy to conform to the surface of the MALDI target. During sample preparation, the concentrator is sealed on the MALDI target and sample solutions are deposited in the wells of the concentrator (see Figure 1C). Figure 2 shows that the MALDI sample concentrators are designed to match with the formats of different types of MALDI (21) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). Anal. Chem. 1998, 70, 4974-4984. (22) Anderson, J. R.; Chiu, D. T.; Jackman, R. J.; Cherniavskaya, O.; McDonald, J. C.; Wu, H.; Whitesides, S. H.; Whitesides, G. M. Fabrication of Topologically Complex Three-Dimensional Microfluidic Systems in PDMS by Rapid Prototyping. Anal. Chem. 2000, 72, 3158-3164. (23) Chen, X.; Wu, H.; Mao, C.; Whitesides, G. M. A Prototype Two-Dimensional Capillary Electrophoresis System Fabricated in Poly(dimethylsiloxane). Anal. Chem. 2002, 74, 1772-1778.

Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

6161

Figure 1. Illustrations of the design of the matrix-assisted laser desorption/ionization (MALDI) sample concentrator: (A) illustration of a six-well MALDI sample concentrator, (B) illustration of a MALDI target (the wells of the MALDI sample concentrator match the predefined spot positions on the MALDI target), and (C) a crosssectional view of the MALDI sample concentrator sealed on a MALDI target. Three wells filled with liquid are illustrated.

Figure 2. Examples of MALDI sample concentrators and their corresponding MALDI target: (A) an Applied Biosystems 100-position MALDI target, (B) a 100-well MALDI sample concentrator sealed on the 100-position MALDI target, (C) a Bruker microplate-size MALDI target, and (D) a 96-well MALDI sample concentrator sealed on the microplate-size MALDI target.

targets. For the 100-spot MALDI target (Figure 2A) used in the Applied Biosystems’ Voyager matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometers, a 100well MALDI sample concentrator (Figure 2B) is designed to match with its size and spot positions. For the microplate-size MALDI targets (Figure 2C) used in Bruker Daltonics’ MALDI mass spectrometers, a 96-well MALDI sample concentrator (Figure 2D) is designed to match with its size and 96 of its 384 spot positions. When the MALDI sample concentrator was used in the experiment, it was first aligned and placed on the MALDI target. The seal was further strengthened by hand, using even pressure across the concentrator. Hand pressure is sufficient to remove any air bubbles that may be trapped between the bottom surface of the concentrator and the surface of the MALDI target. When the MALDI sample concentrator was tightly sealed on the MALDI target, liquids were filled into the wells of the concentrator without leakage. The assembly was centrifuged to facilitate evaporation of the solvents (details described in the next section). During centrifugation, the sealing between the concentrator and the MALDI target is further strengthened, because of the centrifugal 6162

Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

force and the elastomeric property of the concentrator. After multiple steps of sample processing, the concentrator was removed from the MALDI target by carefully lifting the edge and peeling from the target. Evaporation of Solvents in the Wells of the MALDI Sample Concentrator. Solvents in the wells of the concentrator can be evaporated by placing the concentrator-target system in a vented area or by centrifuging the concentrator-target system. For the evaporation of a relatively large volume (>10 µL) of solvents, centrifugation is more efficient to accelerate evaporation and ensure the focusing of the solutes to the bottom of the wells. Two forms of centrifugation can be used: centrifugation under atmospheric pressure and centrifugation under vacuum. When centrifugation under atmospheric pressure is used, evaporation of the solvents is accelerated by convection that is created during centrifugation. When centrifugation under vacuum is used, evaporation of the solvents is accelerated by the vacuum. In the experiments described in this paper, sample evaporations were performed using an Eppendorf Vacufuge Concentrator, Model 5301. The centrifuge is equipped with a rotor that can carry microplates. When a microplate-size MALDI target was used, the concentrator-MALDI target assembly was directly placed on the microplate carrier of the centrifuge rotor. When the Applied Biosystems 100-position MALDI target was used, a microplatesized holder (BD Biosciences Discovery Labware, Bedford, MA) was used to hold the concentrator-MALDI target assembly. The holder was placed on the microplate carrier of the centrifuge rotor. The centrifugation speed is at a default setting of 1400 rpm, the temperature is set at room temperature, and the vacuum pressure is ∼0.5 mbar (∼0.4 Torr). The speed of evaporation has been determined to be highly dependent on the type and amount of the solvents, and the geometry of the concentrator wells. Evaporation of solutions in larger wells has been determined to be much quicker than in smaller wells. When a vacuum centrifuge is used for evaporation, the evaporation time is also highly variable, depending on the vacuum used. With our experimental setup, 3-4 h are required to completely dry 50 µL per well of aqueous solutions, 30-40 min are required to completely dry 50 µL per well of 90% acetonitrile solutions, and 10-15 min are required to completely dry 10 µL per well of matrix solutions (in 75% acetonitrile) in the 100-well MALDI sample concentrator. Desalting, Concentrating, and Spotting Samples Using C18 Resin-Packed Pipet Tips. The following protocol was used to desalt, concentrate, and spot samples with the C18 resin-packed pipet tips: (1) A fresh C18 resin-packed pipet tip was first wetted with 100% acetonitrile by repeating the aspirate/dispense steps three times. (2) It was equilibrated with 0.1% TFA in Milli-Q-grade water by repeating the aspirate/dispense steps three times. (3) It was used to bind peptides and/or proteins in the samples by repeating the aspirate/dispense steps of the sample solution 10 times. (4) It was washed with 0.1% TFA in Milli-Q-grade water by repeating the aspirate/dispense steps three times. At the final aspirate/dispense cycle, the dispensing part was delayed until the next step was performed, so that the pipettor with the C18 resin-

Figure 3. Pictures and illustrations of a concentrating and spotting experiment: (A) a MALDI sample concentrator is sealed on a MALDI target, and a cytochrome c solution (1 mg/mL in 75% acetonitrile) has been added into each well; (B) after evaporating the solvents by centrifugation, the cytochrome c solution is concentrated to the bottom of the wells; (C) when the cytochrome c solution in each well is completely dried, the MALDI sample concentrator is removed from the MALDI target, exposing an array of cytochrome c sample spots; and (D) illustrations of the MALDI sample concentrator and MALDI target corresponding to the photographs in panels A, B, and C. Red dots represent solutes and yellow background represents solvent.

packed pipet tip can be put aside for a moment without drying the C18 resin. (5) An elution solutions1 µL of 0.1% TFA/50% acetonitrile with 10 mg/mL of matrix (R-cyano or sinapinic acid)swas dispensed on the MALDI target using another pipettor with a standard pipet tip. (6) After finishing the last dispensing cycle of step 4, the peptides and/or proteins bound in the C18 resin-packed pipet tip were eluted into the elution solution on the MALDI target by repeating the aspirate/dispense steps three times. (7) Finally, the sample droplet on the MALDI target was allowed to dry in ambient air. Desalting, Concentrating, and Spotting Samples Using the MALDI Sample Concentrator. The following protocol was used to desalt, concentrate, and spot samples with the MALDI sample concentrator: (1) A MALDI sample concentrator was sealed on the MALDI target. (2) The sample solutions were added to each well of the MALDI sample concentrator. The concentrator-MALDI target assembly was centrifuged until all samples were completely dried. (3) Each well containing a dried sample was washed with a washing solutions0.1% TFA in Milli-Q grade watersby repeating the dispense/aspirate cycle of the washing solution in the wells two times. The volume of the washing solution used per well was the same as the volume of samples added per well in step 2. (4) An elution solutions0.1% TFA/90% acetonitrileswas added to each well of the MALDI sample concentrator. The volume of elution solution added per well was the same as the volume of samples added per well in step 2. The concentrator-MALDI target assembly was centrifuged until all samples were completely dried. (5) A matrix solutions10 µL per well of 0.5 mg/mL R-cyano (or 1 mg/mL sinapinic acid) in 75% acetonitrileswas added to each well of the MALDI sample concentrator. The concentratorMALDI target assembly was centrifuged until all samples were completely dried. (6) Finally, the MALDI sample concentrator was removed from the MALDI target to expose the array of sample spots.

Trypsin Digestion of Proteins in Solution Followed by Desalting, Concentrating, and Spotting Digestion Mixtures Using C18 Resin-Packed Pipet Tips. To perform trypsin digestion of proteins in solution, the proteins were first dissolved in the digestion buffer: 25 mM ammonium bicarbonate. A 10fold trypsin stock solution then was added to the digestion buffer to obtain a final trypsin concentration of 10 ng/µL. The digestion mixture was then incubated at 37 °C for 1 h. The digestion mixture then was desalted, concentrated, and spotted using a C18 resinpacked pipet tip, following steps 1-7 described in a previous section, “Desalting, Concentrating, and Spotting Samples Using C18 Resin-Packed Pipet Tips”. In-Well Trypsin Digestion of Proteins and Desalting, Concentrating and Spotting Digestion Mixtures Using the MALDI Sample Concentrator. The following protocol was used to perform in-well trypsin digestion and then desalt, concentrate, and spot the digestion mixtures with the MALDI sample concentrator: (1) A MALDI sample concentrator was sealed on the MALDI target. (2) The protein sample solutions were added to each well of the MALDI sample concentrator. The concentrator-MALDI target assembly was centrifuged until all samples were completely dried. (3) Freshly prepared trypsin solutions10 ng/µL trypsin in 25 mM ammonium bicarbonateswas added to each well that contained a dried protein sample. The volume of trypsin solution used per well was the same as the volume of the protein samples added per well in step 2. The concentrator-MALDI target assembly was then incubated at 37 °C for 1 h. At the end of the 1 h incubation, the assembly was centrifuged until all samples were completely dried. The samples then were washed and concentrated on the MALDI target, following steps 3-6 described in a previous section, “Desalting, Concentrating, and Spotting Samples Using the MALDI Sample Concentrator”. Mass Spectrometry. MALDI-TOF mass spectra were acquired on a Voyager-DE MALDI-TOF mass spectrometer (PerSeptive Biosystems/Applied Biosystems, Framingham, MA). Mass spectra were recorded in the positive-ion, delayed-extraction (DE) Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

6163

mode. All spectra were acquired with an accelerating voltage of 20 kV, 95% grid voltage, 0.1% guide wire, a delayed extraction time of 350 ns, and a low-mass gate of 500 Da. Each spectrum obtained was a sum of ∼100 laser shots. The instrument was externally calibrated on a mixture of human angiotensin II (molecular weight (MW) of 1046.54), synthetic peptide P14R (MW ) 1533.86), human ACTH fragment 18-39 (MW ) 2465.20), bovine insulin oxidized B chain (MW ) 3494.65), and bovine insulin (MW ) 5730.61). Mass spectra were analyzed in the Voyager-DE’s Data Explorer software. Advanced baseline correction was performed for all spectra using the following parameters: peak width, 32; flexibility, 0.5; and degree, 0.1. RESULTS AND DISCUSSION Concentrating Samples on a MALDI Target. In the first set of proof of principle experiments, a solution of cytochrome c (1 mg/mL in 75% acetonitrile) was used to test the ability of the MALDI sample concentrator to concentrate samples on the MALDI target without causing cross contaminations between neighboring wells. Figure 3 shows the photographs and a schematic presentation of this example sample concentrating procedure. In the first step, the concentrator was sealed with the MALDI target and sample solutions were added to the wells (see Figure 3A). The concentrator-MALDI target assembly then was centrifuged to accelerate solvent evaporation. As the solvents evaporated, the samples were concentrated to the bottom of the wells (see Figure 3B). After the sample had completely dried, the concentrator was peeled off the MALDI target (Figure 3C), leaving an array of sample spots on the MALDI target. In these experiments, the high concentration cytochrome c solution was used for visualization purposes and 75% acetonitrile was used to minimize the protein binding to the surface of the concentrator wells (to be discussed below). The results of these experiments demonstrated that the MALDI sample concentrator can be used to allow large volume of samples to be deposited directly on MALDI target and concentrated to dried sample spots without cross contamination of neighboring wells. It was found that if an aqueous solution of proteins or peptides was deposited in the concentrator wells and dried in a centrifuge, the proteins and peptides would bind to the hydrophobic surface of the concentrator wells as the solution dried. However, if a solution with high organic solvent content, such as 90% acetonitrile, was deposited in the concentrator wells and dried in a centrifuge, the proteins and peptides would be concentrated to the bottom of the wells and dried on MALDI target without binding to the hydrophobic surface. These findings are supported by the following results. In the experiments described in the following section (Figures 4, 5, and 6), if the elution step (the elution solution contains 90% acetonitrile) was skipped, the resulting spectra usually contain no peaks of the peptides, indicating that the peptides were bound on the hydrophobic surface of the concentrator wells when the aqueous solutions dried in the wells. Therefore, to concentrate a sample in aqueous solution efficiently using the MALDI sample concentrator, two drying steps must be used. The first step dries the samples in aqueous solution and leaves the proteins and peptides binding to the hydrophobic surface in the concentrator wells. A solution that contains organic solvent, such as 90% acetonitrile, then is added to the same wells 6164 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

Figure 4. Illustrations of a method for desalting, concentrating, and spotting samples on a MALDI target. Red dots represent large molecule solutes (such as peptides or proteins), and green dots represent small molecule solutes (such as salts); the blue background represents water, and the yellow background represents a solvent with high organic contents. (A) A MALDI sample concentrator is sealed on a MALDI target and sample solutions are added to each well. The sample solutions are completely dried during centrifugation. As the sample solutions are drying, the solutes are deposited on the hydrophobic surface inside each well. (B) A washing solution is added to each well. The large molecule solutes such as peptides and proteins are bound on the hydrophobic surface, whereas the small molecule solutes such as salts go back into the washing solution. The solutions are then aspirated, leaving the molecules bound on the hydrophobic surface. (C) A solvent with high organic content is added to each well. The molecules bound on the hydrophobic surface will dissociate from the surface and go into the solvent. The solution is again dried during centrifugation. This time, the solvent keeps the large molecule solutes from binding to the hydrophobic surface during this process. (D) After the solution is dried, a small volume of matrix solution (not shown) is added to each well and the solution is dried again. This final drying step creates sample spots that contain matrix crystals. The MALDI sample concentrator is then removed to expose the sample spots on the MALDI target.

and allowed to dry. The second drying step concentrates the samples to the bottom of the wells, which is the surface of the MALDI target. These processes will be further explained in details in the next section and in Figure 4. Desalting and Concentrating Samples on a MALDI Target. Because the proteins and peptides bind to the hydrophobic surface of the concentrator wells when an aqueous solution of proteins or peptides is dried in the wells, it is possible to use the surface as a reverse phase chromatography media for purifying and desalting peptide or protein samples. Figure 4 describes a method for purifying and desalting samples using the MALDI sample concentrator. In the first step (Figure 4A), the samples are added to the wells of the concentrator (attached to the MALDI target) and

allowed to evaporate during centrifugation. The solutes in the sample solution are deposited on the surface of the concentrator wells. In the second step (Figure 4B), a washing solution is added to the wells to dissolve the salts contained in the dried sample. The washing solution is then aspirated and discarded while the surface bound proteins or peptides remain in the wells. In the third step (Figure 4C), an elution solution is added to the wells. The peptides or proteins are dissociated from the well surface and dissolved into the elution solution. As the solvents of the elution solution are evaporated during centrifugation, the peptides or proteins are focused to the bottom of the wells. A matrix solution is then added into the wells and allowed to dry. In the final step (Figure 4D), after the matrix crystallizes, the concentrator is detached to expose the sample spots for MALDI MS analysis. To demonstrate the effectiveness of this method in desalting and concentrating peptide samples, solutions of a mixture of five standard peptides (including angiotensin II, synthetic peptide P14R, ACTH fragment 18-39, bovine insulin oxidized B chain, and bovine insulin, with different concentrations in 25 mM ammonium bicarbonate buffer) were processed and studied by MALDI-TOF MS. Two sample preparation methods were compared: (i) 50 µL of the peptide solutions were desalted, concentrated, and spotted using C18 resin-packed pipet tips; and (ii) 50 µL of the same peptide solutions were desalted, concentrated, and spotted using the MALDI sample concentrator, following the schemes in Figure 4. Figure 5A compares the mass spectra obtained from 100 nM, 50 nM, 20 nM, and 10 nM peptide solutions, using the two sample preparation methods. Peaks from the five peptides were indicated with numbers. The results indicated that, compared to using C18 resin-packed pipet tips, the recovery of low-concentration peptides is improved using the new method. Figure 5B shows higherresolution spectra that contain two peaks, from the synthetic peptide P14R and ACTH fragment 18-39, obtained from 2 nM, 1 nM, and 0.5 nM peptide solutions, using the new method. At these concentrations, we found that the spectra obtained using C18 resin-packed pipet tips do not contain the peaks of the peptides. These results further demonstrate that the new method improves the ability of recovering peptides from low-concentration solutions. To further demonstrate the effectiveness of the new desalting and concentrating method, solutions of trypsin digestion mixtures of BSA in 25 mM ammonium bicarbonate buffer, with different concentrations (C) and volumes (V) but same total amount of material (equal to the product CV), were processed and studied by MALDI-TOF MS. Figure 6 compares the mass spectra obtained from different sample preparation methods. In Figure 6A, 0.5 µL of a 1000 nM BSA digest solution was mixed with 0.5 µL of matrix solution and spotted directly on the MALDI target. At this high concentration, sample desalting and concentrating are not necessary to obtain a good spectrum. In Figure 6B, 1 µL of a 500 nM BSA digest solution was desalted and spotted using a C18 resin-packed pipet tip. The spectrum closely resembles the spectrum in Figure 6A, indicating a good recovery of peptides from sample processing. In Figure 6C, 10 µL of a 50 nM BSA digest solution was desalted, concentrated, and spotted using a C18 resin-packed pipet tip. The spectrum indicates that some peptides are lost during sample processing. In Figure 6D, 50 µL of a 10 nM BSA digest solution was desalted, concentrated, and

Figure 5. (A) Comparison of the mass spectra of five standard peptides obtained using two sample preparation methods. In the left column, 50 µL of samples at each concentration (100 nM, 50 nM, 20 nM, and 10 nM) were desalted, concentrated, and spotted using C18 resin-packed pipet tips. In the right column, 50 µL of samples at each concentration (100 nM, 50 nM, 20 nM, and 10 nM) were desalted, concentrated, and spotted using the MALDI sample concentrator, following the scheme shown in Figure 4. The labeled peaks correspond to (1) angiotensin II, (2) synthetic peptide P14R, (3) ACTH fragment 18-39, (4) bovine insulin oxidized B chain, and (5) bovine insulin. (B) Higher-resolution spectra containing two peaks from the synthetic peptide P14R and ACTH fragment 18-39, obtained by desalting, concentrating, and spotting 50 µL of 2 nM, 1 nM, and 0.5 nM peptide solutions using the MALDI sample concentrator, following the scheme shown in Figure 4.

spotted using a C18 resin-packed pipet tip. Most of the peptide peaks are lost, presumably because of the poor recovery in the desalting process. In Figure 6E, 50 µL of a 10 nM BSA digest solution was desalted, concentrated and spotted using the MALDI Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

6165

Figure 7. Illustrations of a method for binding and digesting proteins using the MALDI sample concentrator sealed on a MALDI target: (A) protein solutions are added to the wells and dried by centrifugation (the red dots represent proteins), and the proteins are immobilized on the hydrophobic surface as the solutions dry; (B) trypsin solution is added to the wells (the blue diamonds represent trypsin, and the small red dots represent the digestion products of proteins). Table 1. List of Identified Peaks in Figure 8

Figure 6. Comparison of the mass spectra of bovine serum albumin (BSA) trypsin digestion samples obtained by different preparation methods: (A) 0.5 µL of a 1000 nM BSA digest solution was mixed with 0.5 µL of matrix solution and spotted directly on the MALDI target; (B) 1 µL of a 500 nM BSA digest solution was desalted and spotted using a C18 resin-packed pipet tip; (C) 10 µL of a 50 nM BSA digest solution was desalted, concentrated, and spotted using a C18 resinpacked pipet tip; (D) 50 µL of a 10 nM BSA digest solution was desalted, concentrated, and spotted using a C18 resin-packed pipet tip; and (E) 50 µL of a 10 nM BSA trypsin digestion solution was desalted, concentrated, and spotted using the MALDI sample concentrator following the scheme shown in Figure 4.

sample concentrator, following the schemes in Figure 4. The spectrum closely resembles the spectrum in Figure 6A, showing an improved peptide recovery in the desalting and concentrating process. The spectra in Figure 6B-D suggest that, as the sample concentration becomes smaller, it is more difficult to use a C18 resin-packed pipet tip to pick up all the peptides in the solution. Similar to other reverse phase column purification methods, the C18 resin-packed pipet tip relies on the hydrophobic surface of the resin to capture the peptides as the solution is flowing through. At low concentration, a larger proportion of peptides will flow through without binding to the hydrophobic surface. The spectrum in Figure 6E shows remarkable improvement of peptide recovery at low concentration using the new desalting method outline in Figure 4, in which the low concentration solution is dried on the hydrophobic surface. During the drying process, the 6166 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

peaka

mass

sequenceb

peaka

mass

sequenceb

1 2 3 4 5 6 7 8 9 10

1087.6 1126.6 1149.7 1171.7 1274.7 1314.7 1378.7 1529.7 1669.9 1720.0

R 91-99 β 96-104 β 133-144 R 1-11 β 31-40 β 18-30 β 121-132 R 17-31 β 67-82 β 105-120

11 12 13 14 15 16 17 18 19

1798.0 1833.9 2058.9 2228.2 2529.2 2996.5 3124.6 3314.7 4201.3

β 66-82 R 41-56 β 41-59 β 9-30 β 83-104 R 62-90 R 61-90 β 31-59 R 100-139

a These numbers correspond to the numbers that appear in the mass spectra. b The sequences were obtained using the PeptideMass tool on the ExPASy website (http://us.expasy.org/tools/ peptide-mass.html). The maximum number of missed cleavages is set at 1. R denotes the R chain, and β denotes the β chain of human hemoglobin.

concentration increases and the peptides are driven toward the hydrophobic surfaces. This will help increase the proportion of peptides binding to the hydrophobic surface, compared to the conventional flow-through method. Trypsin Digestion, Desalting, and Concentrating Samples on a MALDI Target. Because the proteins bind to the hydrophobic surface of the concentrator wells when an aqueous solution of proteins is dried in the wells, it is possible to use the surface to immobilize proteins for trypsin digestion in the wells. The drying process will cause protein denaturing, which may increase the efficiency of trypsin digestion. Figure 7 describes a method of immobilizing proteins and performing trypsin digestion in the wells of the MALDI sample concentrator. In the first step (Figure 7A), the protein samples are added to the wells of the concentrator (attached to the MALDI target) and allowed to evaporate by centrifugation. The protein samples are dried and the proteins are bound to the hydrophobic surface of the concentrator wells. In the second step (Figure 7B), a trypsin solution is added to the wells containing the bound proteins. The concentrator-MALDI target assembly is then incubated at 37 °C for 1 h. After incubation, the trypsin digestion mixtures are

Figure 8. Comparison of mass spectra of the trypsin digestion products of human hemoglobin obtained by different methods. For each method, two spectra were obtained: one spectrum was obtained using an R-cyano matrix (A and C, spectra in the region of m/z ) 1000-2000) and another spectrum was obtained using sinapinic acid (B and D, spectra in the region of m/z ) 2000-5000). The spectra in panels A and B were obtained via in-solution digestion of 500 ng of hemoglobin, followed by desalting, concentrating, and spotting using C18 resin-packed pipet tips. The spectra in panels C and D were obtained by in-well digestion of 500 ng of hemoglobin, using the method shown in Figure 7, followed by desalting, concentrating, and spotting, using the method shown in Figure 4. The spectrum in panel E is the indicated portion of the spectrum in panel A without baseline corrections, and the spectrum in panel F is the indicated portion of the spectrum in panel C without baseline corrections.

desalted and concentrated using the method described in Figure 4. To demonstrate the effectiveness of this method in obtaining a peptide mass fingerprinting map, 500 ng of human hemoglobin either was digested in solution and desalted, concentrated, and spotted using a C18 resin-packed pipet tip, or was digested inwell and desalted, concentrated, and spotted using a MALDI sample concentrator. Figure 8 compares the mass spectra obtained from these two methods. For each method, two spectra were obtained: one spectrum was obtained using an R-cyano matrix (spectra in Figure 8A and C, m/z ) 1000-2000) and another spectrum was obtained using sinapinic acid (spectra in Figure 8B and D, m/z ) 2000-5000). The mass spectra in Figure 8A and B were obtained by digesting 500 ng of hemoglobin in 10 µL of trypsin solution. The digestion mixture was then desalted, concentrated, and spotted using C18 resin-packed pipet tips. Mass spectra in Figure 8C and D were obtained by digesting 500 ng of

hemoglobin using the method described in Figure 7. The hemoglobin solution was first dried in the wells of the MALDI sample concentrator. Then, 10 µL of trypsin solution was added to the well for digestion. The digestion mixture was then dried, desalted, concentrated, and spotted following the schemes in Figure 4. The identified peaks and the matched sequences are shown in Table 1. The sequence coverage obtained by the insolution digestion/pipet tip method is 32%, whereas the sequence coverage obtained by the MALDI sample concentrator in-well digestion method is 50%. The difference appears mostly in the higher-mass region (m/z ) 2000-5000), where the new method has resulted in more identified peaks. Figures 8E and 8F represent magnified views of the spectra shown in Figures 8A and 8C in the m/z region of 1600-1800 (without baseline corrections). The spectrum in Figure 8F contains peaks 9 and 10 (Table 1) and their respective sodium adduct, whereas the spectrum in Figure 8E contains only peak 9. This comparison shows that the signal-toAnalytical Chemistry, Vol. 78, No. 17, September 1, 2006

6167

noise ratio is improved with the use of the MALDI sample concentrator. The improvement of sequence coverage may be attributed to two factors: (i) the proteins were immobilized on the hydrophobic surface of the concentrator well for digestion, as opposed to being in solution for digestion, and (ii) the desalting and concentrating process was performed using the method described in Figure 4, as opposed to using the C18 resin-packed pipet tip method. When only one of these factors was utilized (results not shown), the improvement of sequence coverage was determined to be less than that shown in Figure 8. This suggests that both factors contributed to the improvement of sequence coverage. Further studies are underway to better understand the contributions from each individual factor. CONCLUSIONS We have demonstrated an array of new methods for matrixassisted laser desorption/ionization (MALDI) sample preparation using the MALDI sample concentrator. The principal foci of this work are on (i) the ability of depositing and processing relatively large volume of samples directly on MALDI target, (ii) the effectiveness of a new desalting method in recovering lowconcentration peptides, and (iii) the ability of immobilizing proteins on the hydrophobic surface for in-well trypsin digestion. (24) McComb, M. E.; Perlman, D. H.; Huang, H.; Budnik, B. A.; Kaur, P.; O’Connor, P. B.; Costello, C. E. Direct Protein 2D-LC MALDI with On-Target Digestion for High-throughput Proteomics Analyses. Presented at the 53rd ASMS Conference, Poster No. ThP553, 2005. (25) Snovida, S. I.; Chen, V. C.; Perreault, H. Anal. Chem., accepted for publication.

6168

Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

It has been demonstrated that the MALDI sample concentrator is a useful tool for depositing a relatively large volume of samples directly on the MALDI target for a series of sample processing that results in prepared sample spots. The new sample desalting, concentrating, and spotting method yields a better recovery of low-concentration peptides, compared to the conventional method using C18 resin-packed pipet tips. The new in-well trypsin digestion method yields higher sequence coverage than the conventional in-solution digestion combined with desalting and concentrating using C18 resin-packed pipet tips. The reduction of sample transferring steps and the method of drying lowconcentration samples on a hydrophobic surface are keys to the improvement of sample recovery. We are continuing studies to further understand the potentials and limitations of the new methods. In parallel to this work, sample preparation using the MALDI sample concentrator has been explored in other laboratories.24,25 ACKNOWLEDGMENT We thank Dr. Mark E. McComb and Dr. David Perlman of the Cardiovascular Proteomics Center; Boston University School of Medicine for numerous help in this work. We also thank Dr. Catherine E. Costello of Boston University School of Medicine for helpful discussions. Finally, we thank Dr. Charles L. Crespi of BD Biosciences Discovery Labware for tremendous support for this work. Received for review February 15, 2006. Accepted June 19, 2006. AC060286B