High-Throughput Peptide Mass Mapping Using a Microdevice Containing Trypsin Immobilized on a Porous Polymer Monolith Coupled to MALDI TOF and ESI TOF Mass Spectrometers Dominic S. Peterson,† Thomas Rohr,† Frantisek Svec,†,‡ and Jean M. J. Fre´ chet*,†,‡ E. O. Lawrence Berkeley National Laboratory, Materials Sciences Division, Berkeley, California 94720, and Center for New Directions in Organic Synthesis,§ Department of Chemistry, University of California, Berkeley, California 94720-1460 Received July 4, 2002
Abstract: An enzymatic microreactor with a volume of 470 nL has been prepared by immobilizing trypsin on a 10 cm long reactive porous polymer monolith located in a 100 µm i.d. fused silica capillary. This reactor affords suitable degrees of digestion of proteins even after very short residence times of less than 1 min. The performance is demonstrated with the digestion of eight proteins ranging in molecular mass from 2848 to 77 754. The digests were analyzed using mass spectrometry in two modes: off-line MALDI and in-line nanoelectrospray ionization. The large numbers of identified peptides enable a high degree of sequence coverage and positive identification of the proteins. The extent of sequence coverage decreases as the molecular mass of the digested protein increases. Keywords: high-throughput protein identification • immobilized enzyme • mass spectrometry • monolith • proteomics
Introduction Despite significant advances in the analytical methodologies applicable to proteomics,1-3 methods and devices that provide significant improvements in analytical throughput are still highly desirable. The use of mass spectrometry has added a new dimension to the analysis of proteins, but since a measured mass can correspond to numerous different molecules, the determination of mass alone is not sufficient for characterization, and more precise methods are necessary to map protein composition. These methods typically involve the digestion of the protein of interest by a proteolytic enzyme followed by identification of the resulting peptides using mass spectrometry. This approach, coupled to an understanding of the cleavage process, affords a peptide map that is unique for each protein and allows its identification by searching existing databases. Since the amounts of proteins available for analysis are typically small, miniaturization of the mapping procedures is also very important.4-8 †
E. O. Lawrence Berkeley National Laboratory. ‡ University of California. § The Center for New Directions in Organic Synthesis is supported by Bristol-Myers Squibb as Sponsoring Member. 10.1021/pr0255452 CCC: $22.00
2002 American Chemical Society
The most common current protocol for protein digestion utilizes trypsin in solution. The typical reaction time is 24 h since the trypsin-to-substrate ratio has to be kept very low (1: 50) to avoid excessive autodigestion of trypsin and the resulting formation of additional peptide fragments derived from the proteolytic enzyme itself. Obviously, the use of higher trypsin concentrations would accelerate the digestion as demonstrated by Lazar et al.9 in a microfluidic analytical format. In their work, mixtures with much higher trypsin-to-protein ratios (20:11:1) were prepared in a vial, followed by a digestion period of 2-15 min and subsequent transfer to a nanoESI microchip for TOF-MS detection. Indeed, the digestion was much faster and the short digestion time was sufficient to achieve a sequence coverage as high as 100% for glucagon (a peptide with 29 amino acid residues) and 95% for cytochrome C. As expected, a strong background due to ions originating from autodigested trypsin was also observed. In an alternative approach, the same group mixed trypsin and insulin B-chain on the chip then allowed the digestion to proceed for up to 15 min prior to performing an electrophoretic separation of the peptides followed by detection using laser-induced fluorescence.10 The undesired autodigestion can be eliminated by immobilizing the enzyme on the solid support that isolates the enzyme moieties.11-14 For example, Blackburn and Anderegg have used a capillary reactor packed with Poroszyme beads with high enzymatic activity to carry out off-line digestion for 10 min prior to collecting the peptide solution in vials, performing a desalting operation and finally injecting in an ESI-MS instrument.15 The “on-chip” implementation of packed immobilized enzyme reactor is more difficult to achieve. L’Hostis et al.16 have placed functionalized controlled pore glass beads bearing an immobilized enzyme in a 2.25 µL chamber fabricated in a silicon substrate. Wang et al.17 have implemented a system consisting of a glass chip with a 1.8 µL cavity filled with trypsin immobilized on agarose beads and an electrophoretic separation channel connected to nanoESI-MS detection. After a residence time of 3-6 min, this device digested melittin and cytochrome c with a sequence coverage of 100 and 92%, respectively. To avoid micromanipulation with beads, several groups have immobilized enzymes directly on the walls of microchannels.18-21 Since the surface-to-volume ratio of a microchannel is small, the overall enzymatic activity of the device is limited. An Journal of Proteome Research 2002, 1, 563-568
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technical notes
High-Throughput Peptide Mass Mapping Using a Microdevice
increase in surface area can be achieved by fabricating a layer of porous silicon on the walls of a microchannel.22-24 However, the amount of protein that can be immobilized remains small and activity is low. Thus, only 11 peptides have been identified in the MALDI TOF mass spectrum of casein after 12 s of digestion in the optimized device.24 We have demonstrated previously the very facile on-chip preparation of porous polymer monolith using photoinitiated polymerization. This photolithographic technique involving UV irradiation through a mask enables the polymerization to be performed only in selected areas of the microdevice.25-28 For example, we integrated monoliths containing azlactone functionalities in various microdevices and used them for enzyme immobilization. The azlactone moieties of the functionalized monoliths react rapidly with proteins,29 thereby immobilizing them on the pore surface. Very high proteolytic activity of immobilized trypsin permitted digestion of model protein myoglobin in less than 12 s. Using MALDI TOF MS analysis of the digest, 102 out of 153 possible amino acids in peptide fragments were identified giving a sequence coverage of 67%.28 In this paper, we report the performance of a similar flowthrough enzymatic microreactor located within a capillary microchannel. This reactor contains a porous polymer monolith with immobilized trypsin and is used for the rapid digestion of a variety of proteins followed by off-line MALDI TOF MS or in-line ESI TOF MS detection of the peptides.
Experimental Section Materials. Trypsin (bovine pancreas), melittin (bee venom), myoglobin (equine heart), cytochrome c (equine heart), ribonuclease A (bovine pancreas), holo-transferrin (bovine), bovine serum albumin (BSA), R1-acid glycoprotein (bovine serum), and casein (bovine milk) were obtained from Sigma (St. Louis, MO). 1-Vinyl-4,4-dimethylazlactone (VAL) was a generous gift from the 3M Co. (St. Paul, MN). Most of the other compounds were purchased form Aldrich (Milwaukee, WI). Teflon-coated fused silica capillary was from Polymicro Technologies (Phoenix, AZ). Monolithic Supports. The internal surface of Teflon-coated 100 µm i.d. fused silica capillary was washed with acetone and water, activated with a 0.2 mol/L sodium hydroxide solution for 30 min, washed consecutively with water, 0.2 mol/L HCl for 30 min, and then with water and acetone, and dried in a stream of nitrogen. The capillary was then filled with a 30% solution of 3-(trimethoxysilyl)propyl methacrylate in acetone, both ends sealed, and kept at room temperature for 24 h to achieve surface vinylization and enable covalent attachment of the monolith to the wall.30 The capillary was then washed with acetone and dried, and the sections where photopolymerization was undesirable were covered with an opaque mask produced by painting the surface with a flat black fast dry enamel. The length of the unmasked part was kept at 100 mm. To prepare the monolith, the capillary was filled completely with a previously optimized polymerization mixture28 consisting of monomers (20% ethylene dimethacrylate, 8% 1-vinyl-4,4dimethylazalactone, and 12% 2-hydroxyethyl methacrylate), porogenic solvent (60% 1-decanol), and a photoinitiator (2,2dimethoxy-2-phenylacetophenone, 1% with respect to monomer). The capillary was exposed to UV irradiation (Oriel deep UV illumination series 8700 fitted with a 500 W HgXe lamp, Stratford, CT) for 6 min to produce monolith with a median 564
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pore diameter of 1.02 µm, a pore volume of 1.33 mL/g, a specific surface area of 6.2 m2/g, and a porosity of 51.9%.27 Immobilization of Trypsin. A procedure developed previously was used to immobilize trypsin on the monoliths.28 Trypsin (2 mg/mL) was dissolved in an aqueous solution containing 0.5 mol/L of sodium sulfate, 0.1 mol/L of sodium carbonate, and 0.05 mol/L of benzamide. This solution was pumped through the monolith for 3 h. The monolith was then washed for 1 h with 1 mol/L of aqueous ethanolamine to quench the unreacted azlactone functionalities and equilibrated in a 50 mmol/L TRIS buffer, pH 8.0, for 12 h using a KD Scientific syringe pump (New Hope, PA). Protein Digestion. Protein solutions (20 ( 2 pmol/µL) in 50 mmol/L of Tris buffer pH 8 were pumped using a KD Scientific syringe pump through the reactor at a flow rate of 0.5 µL/min affording a residence time of 57 s. Probot microfraction collector (LC Packings, San Francisco, CA) automatically combined the effluent with R-cyano-4-hydroxycinnamic acid (CHCA) matrix (0.5 µL/min) and spotted for 30 s onto a 96 × 2 stainless steel plate with a hydrophobic mask plate (Applied Biosystems, Foster City, CA). Each spot contains peptides resulting from about 10 pmol of the original protein. One entire row (12 spots) was spotted for each digested protein and the mass spectra collected using a Voyager DE Biospectrometry Workstation (Applied Biosystems). Alternatively, 20 ( 2 pmol/µL protein solutions in 10 mmol/L ammonium acetate solution, pH 6.7, were pumped through the reactor at a flow rate of 0.4 µL/min affording a residence time of 71 s using a Waters Cap-LC chromatograph (Milford, MA). The reactor was attached using Upchurch (Oak Harbor, WA) microtight fittings to a nanoelectrospray needle (New Objective, Woburn, MA). ESI-MS spectra were obtained using an orthogonal acceleration time-of-flight mass spectrometer Micromass LCT (Manchester, U.K.) equipped with a Picoview nanospray interface (New Objective). Each mass spectrum is derived from 60 summed scans acquired within 1 min. All mass spectra were compared with the theoretically predicted ions using the Protein Prospector protein digestion database (http://prospector.ucsf.edu) to “fingerprint” each digest and determine the sequence coverage.31
Results and Discussion Off-Line Procedure with MALDI-TOF MS Detection. In our previous research,28 we have used a 2 cm long monolithic reactor containing immobilized trypsin that was used at a flow rate of 0.5 µL/min to digest myoglobin and afforded very good sequence coverage after only 11.7 s of residence time in the reactor. However, the use of this reactor together with the instrumentation for automated sample preparation resulted in poor signal-to-noise ratio. Therefore, in this study, we increased the length of the microreactor 5-fold to 10 cm to reach a total reactor volume of 471 nL. This enabled an increase in residence time to 57 s at the same flow rate of 0.5 µL/min. Eight proteins ranging in molecular mass from 2848 to 77 574 were passed thorough this enzymatic reactor, automatically combined with the matrix, and spotted onto a MALDI plate using a robotic interface. Figure 1 shows MALDI-TOF MS spectra of the individual digests. Since 12 spots were always prepared and each spot analyzed, the sequence coverage obtained by MALDI MS varies within a certain range. For example, with cytochrome c, this range is 55-80%. This results from the spot to spot
technical notes
Peterson et al.
Figure 1. MALDI TOF MS spectra of peptides obtained by digestion of eight proteins using monolithic immobilized enzyme microreactor. Conditions: protein concentration of 20 ( 2 pmol/µL in 50 mmol/L Tris buffer pH ) 8.0, flow rate through reactor 0.5 µL/min, eluentmatrix ratio 1:1.
variability rather than from a lack of reproducibility in the digestion process since the sequence coverage calculated from
several mass spectra collected from a single spot remains unchanged. In contrast, variability in sequence coverage is Journal of Proteome Research • Vol. 1, No. 6, 2002 565
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High-Throughput Peptide Mass Mapping Using a Microdevice Table 1. Results of Off-Line Protein Digestion-MALDI-TOF MS protein
MW
pI
sequence coverage, %
amino acids identified
missed cleavages
peptide fragments
melittin cytochrome c ribonuclease A myoglobin casein R-acid glycoprotein bovine serum albumin holo-transferrin
2848 11 702 16 461 16 951 24 529 38 419 69 294 77 754
11.1 9.59 8.93 7.36 4.98 5.30 5.80 6.75
100 79.8 84.0 65.3 50.0 22.3 22.9 21.9
26 83 126 100 107 80 139 154
2.1 2.2 2.3 1.9 1.7 1.1 1.7 2.0
10 9 7 7 6 8 10 9
Table 2. Results of In-Line Protein Digestion-ESI TOF MS protein
MW
pI
sequence coverage, %
amino acids identified
missed cleavages
peptide fragments
melittin cytochrome c ribonuclease A myoglobin casein R-acid glycoprotein bovine serum albumin holo-transferrin
2848 11 702 16 461 16 951 24 529 38 419 69 294 77 754
11.1 9.59 8.93 7.36 4.98 5.30 5.80 6.75
100 74.0 38.0 40.5 30.4 18.7 18.5 12.5
26 77 57 62 65 67 111 88
1.75 1.45 0.38 0.71 1.16 0.70 0.81 0.73
12 11 13 14 12 10 16 11
observed among various spots indicating differences in spot quality. Table 1 shows the highest sequence coverage obtained for each digested protein. An ideal coverage of 100% is achieved for the low molecular mass peptide melittin that includes 26 amino acid residues and its tryptic digest consists of 26 identified peptides. In contrast, a sequence coverage of only 22-23% is obtained for proteins with molecular mass in the range of about 40000-80000 such as R-acid glycoprotein, BSA, and holo-transferrin. The decrease in sequence coverage appears to be inversely related to the molecular mass and correlates with the increased barrier to the desired proteinprotein interaction that is required to cleave the specific sequence. The number of peptide fragments indicates how many peaks were identified in the mass spectrum. A larger number of fragments enables a more reliable match between the database and the data itself. Two additional parameters also describe the efficiency of the protein digestion: (i) the sum of the number of amino acids identified in each fragment, which affords the total number of identified amino acids in peptide fragments, and (ii) the number of missed cleavages. The short time available for the enzymatic reaction to take place results in less than 100% protein digestion efficiency. Thus, some of the identified peptide fragments may still include cleavable amino acid sequences. The number of these missed cleavages is then averaged for all of the identified fragments. On-Line Procedure with ESI MS Detection. Table 2 shows that the sequence coverage obtained for the same series of eight proteins using ESI mass spectra presented in Figure 2 is slightly lower than that observed for MALDI TOF MS. This is due to the use of ammonium acetate buffer for digestion. This buffer is well suited for ESI analysis but its lower pH is not optimal for trypsin. Although the use of more basic buffers such as ammonium bicarbonate for protein digestion followed by ESI MS has been demonstrated, the relative intensity of positively charged ions is always at least 1 order of magnitude lower.9 Under the conditions used in these experiments, the effect of molecular mass is even more evident. Although the sequence coverage for mellitin is again 100% as a result of its small size, it is only 12.5% for the largest protein holo-tranferrin (MW 77 574). It is worth noting that this apparently small number 566
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still corresponds to 88 positively identified amino acids in peptides. In addition, the number of identified peptide fragments is higher than that found using the MALDI analysis and results in a smaller number of missed cleavages.
Conclusions Porous polymer monoliths with reactive azlactone functionalities prepared by UV initiated polymerization at a specific location of the microdevice are well suited for the fabrication of enzymatic microreactors that can be coupled both off-line and in-line with mass spectrometers. Immobilization of trypsin on this support affords an efficient reactor for the rapid digestion of proteins. Compared to the current state-of-theart microfluidic devices for protein digestion that require reaction times of 3-15 min to achieve the desired sequence coverage,9,17,22 our small volume microreactor with monolithic support affords comparable coverage after a residence time of less than 1 min. This makes this reactor very suitable for high throughput protein mapping, one of the most desirable features required to accelerate the research in proteomics. The fast reaction rate clearly results from the rapid mass transfer of the substrate proteins to the reactive sites of immobilized trypsin. In contrast to the diffusional mass transfer of substrates to the enzymes immobilized in porous beads, which is very slow for large proteins, much faster convective transfer is characteristic of the monolithic materials. The protein substrate molecules are transported to the immobilized enzyme moieties by fast flow through the pores of the support.32 Similarly, the products are rapidly removed from the vicinity of the active site of the proteolytic enzyme by the convective flow, thus liberating the site for further reaction. These processes significantly enhance the apparent activity of the enzyme. This effect is most noticeable for large molecules such as biopolymers that have a low intrinsic rate of diffusion. While the proteolytic performance of our microreactor was demonstrated on eight proteins ranging in molecular mass from 2848 to 77 700, there is no reason to doubt this monolithic reactor could be used with a much wider range of proteins.
technical notes
Peterson et al.
Figure 2. ESI TOF MS spectra of peptides obtained by digestion of eight proteins using monolithic immobilized enzyme microreactor. Conditions: protein concentration of 20 ( 2 pmol/µL in 10 mmol/L ammonium acetate buffer pH ) 6.7, flow rate through reactor 0.4 µL/min.
Although the sequence coverage is likely to further decrease as the molecular mass of the digested proteins increases, it is well-known that only three to five fragments are sufficient for the identification of proteins included in today’s databases.33 If necessary, coverage can be increased by using lower flow rates that afford longer residence times and more efficient sample preparation techniques such as off-line vacuum deposition.34 As a result of their high digestion rate, these reactors may also be easily coupled in-line with liquid chromatography or electrochromatography units to enable the separation of proteins and/or peptides. In such coupled system, the microreactor can be placed before the separation column to allow the formation of peptides, followed by their separation (“shot-
gun” proteomics), or the digestion may follow the separation in order to digest the individual proteins or their mixtures containing fewer proteins that the original complex sample. Our approach to a proteolytic microreactor was demonstrated using immobilized trypsin. Obviously, the same technique can be used for wide variety of other enzymes with different specificities and the devices can be used in numerous applications other than protein mapping.
Acknowledgment. This work was supported by the Office of Nonproliferation Research and Engineering of the U.S. Department of Energy under contract No. DE-AC03-76SF00098 and the National Institute of General Medical Sciences, NaJournal of Proteome Research • Vol. 1, No. 6, 2002 567
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High-Throughput Peptide Mass Mapping Using a Microdevice
tional Institutes of Health (GM-48364). We also thank Dr. S. Heilmann and the 3M Company for the gift of vinylazlactone monomer.
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PR0255452
