Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry

Journal of Chromatography A, 1106 (2006) 165–174

Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry Jicheng Duan, Liangliang Sun, Zhen Liang, Jie Zhang, Hui Wang, Lihua Zhang ∗ , Weibing Zhang, Yukui Zhang National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China Available online 20 December 2005

Abstract A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), Nacryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonostrated with the digestion of cytochrome c at the fast flow rate of 1 ␮L/min, which afforded a residence time of 7 s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Furture more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture. © 2005 Elsevier B.V. All rights reserved. Keywords: Monolithic column; Enzymatic reactor; Protein identification; Nano-liquid chromatography-tandem mass spectrometry

1. Introduction With the acceleration of complete genomes discovery, the challenge of proteomics is to identify and characterize proteins encoded by the genomes as efficiently and rapidly as possible [1–3]. Protein identification and characterization is routinely accomplished with mass spectrometry (MS) [2,4]. In most MS approaches, proteins are normally difficult to be directly unambiguously identified to obtain sequence information, because of their relative low efficiency of ionization and fragmentation. Therefore, proteins should be degraded into fragments prior to MS analysis. The most common protocol is accomplished through enzymatic hydrolysis [5]. The proteolytical digestion of protein generally takes place with one or multiple proteases, which usually have their own unique specificities. For example, trypsin is capable of selective

∗

Corresponding author. Tel.: +86 411 84379560; fax: +86 411 84379560. E-mail address: [email protected] (L. Zhang).

0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.11.102

cleaving proteins at arginine and lysine residues, which could provide typically peptides in a mass range compatible with MS for amino acid sequence determination. Therefore, it is often used in proteomic studies [5,6]. The traditional digestions are performed in solution for at least several hours. To avoid the autodigestion of trypsin, which might produce excessive amounts of undesired tryptic fragments and complicate the unambiguous assignment of the studied protein [7], the digestion is generally performed with low concentration enzyme, leading to extended incubation times [8,9]. To solve these problems, immobilized trypsin, which has the advantage of reducing autolysis product formation, allowing the use of higher trypsin concentrations that yield shorter digestion times, has been utilized under various configurations. Furthermore, the immobilized enzyme could be isolated and removed from the protein digests prior to MS easily, eliminating or reducing the influence of the enzyme fragments on MS results. Additionally, the stability of trypsin toward chemical denaturants and organic solvents could be enhanced when immobilized on the solid supports [10].

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In recent years, several reports have demonstrated the feasibility of protein digestion using trypsin immobilized on various supports, such as porous silicon matrix [11–13], glass [14], gel beads [9,15], polymer [16], sol–gel supports [17,18], membrane [19,20] and porous monolithic materials [21–27], among which porous polymer monolithic supports have been recently introduced as novel materials, in which the diffusion resistance during mass transfer has been proved quite small. In addition, this kind of material has the excellent ability to be applied in acid or basic condition. Therefore, they have also been regarded as ideal supports for the immobilization of enzymes and fast conversion of substrates [28–31]. Porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith is one of popular materials that have been used as enzymatic reactor carrier [21,31] because of their biocompatibility and easy modification. However, the activity of epoxide groups towards the amino groups is relatively poor. Therefore, the further modification of the epoxide groups on the supports is usually necessary, resulting in long time preparation process and poor reproducibility. To meet the challenges, some groups applied novel active monomers to prepare the monolithic support for accelerating the enzyme immobilizing. Palm and Novotny [30] had developed a porous polymer monolithic trypsin microreactor for fast peptide mass mapping. The monolith was prepared by the polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and N,N-methylenebisacrylamide in a electrolyte buffer with PEG as molecular template. The polymerization reaction and enzyme immobilization could be completed within 2 h for the high activity of NAS to enzyme. This kind of monolithic reactor offered high flow permeability as well as biocompatibility. However, the content of monomers in the polymerization mixture was restricted by the poor solvability of monomers in aqueous solution, resulting in loose structure and low amount enzyme immobilized [32]. In contrast to the poor solubilizing properties of water towards nonpolar monomers, the alcohol solvent not only allows high amount monomers solvated in the solution, but also has the ability to control the porous properties of the monoliths [32]. The monolithic supports polymerized in a alcohol porogenic solvent system for the immobilization of trypsin were developed by Peterson et al. [23–25], which had good mechanical strength and low back pressure. The azlactone moieties, which could react easily at room temperature with amino or hydrosulfide groups in protein molecules, were incorporated into the monolith by copolymerizing 2-vinly-4,4-dimethylazlactone (VAL) with acrylamide and ethylene dimethacrylate (EDMA). Such materials were prepared in capillaries and chips to couple with MALDI-TOF or ESI-TOF MS for fast protein digestion and peptides mapping. However, the active monomer of VAL now is only used in a few laboratories or companies. Mass spectrometry methods, in particular tandem MS (MS/MS), provide a fundamental set of tools for proteome analysis [1,5,33]. MS/MS spectra of peptides contain not only the information about the mass of peptide but also a pattern of fragment of sequence ions that is fairly unique to a given sequence, creating a higher level of specificity. Because even one peptide

is sufficient to identify a unique protein, this approach is more powerful than the peptide mass mapping where several peptide masses from one protein are used for protein identification. Currently, direct coupling of nanoLC with MS [34,35] or tandem MS [33,36] is the most powerful approach for proteome study. Among all the interfaces, sheathless electrospray and nanospray interfaces are preferred because they have the advantages of high sensitivities, compatibility with low flow rates and little sample dilution. Recently, a novel approach for coupling the liquid-phase separation to mass spectrometer has been suggested by Mazereeuw and coworkers [37,38]. CE-MS with nanospray were performed by adjusting the position of the outlet of the capillary so that the electrical contact was established through the air to the grounded inlet of the heated capillary in MS. Later, Yates and coworkers [39–41] describe an automated method for shotgun proteomics carried out by packing 5 ␮m strong cation exchange resin and reversed phase resin in a biphasic column. The tip at the end of the capillary was tapered and put just in front of the orifice of the heated capillary in mass spectrometer. The spray voltage was applied on the inlet of the capillary to establish the electrical contact without additional spray emitter connected. In this paper, we report the preparation of a monolithic support in an alcohol porogenic solvent system, using NAS as the active monomer to achieve a rigid monolithic structure and high reaction activity towards enzyme. The content of monomers in the polymerization mixture was optimized. The comparative study of enzyme activity was carried out in the newly prepared microreactor and the reactor polymerized in a aqueous buffer using BAEE as substrate. The activities of the immobilized enzyme were characterized by the Michaelis constant (Km ) and the maximum velocity (Vmax ) of the reaction. A length of 17 cm 75 ␮m I.D. capillary with tapered end was packed with 3 ␮m size of reversed phase silica and used as the separation column to directly couple with mass spectrometer without extra spray emitter connected. A high voltage was applied at the inlet of the column to conduct the electrospray potential. The digestion reactions of cytochrome c performed on column and in solution were compared and evaluated using such a nano-LC-MS/MS system. Furthermore, a on-line protein digestion and identification system was developed and applied into the digestion, separation and identification of protein mixture. 2. Experimental 2.1. Chemicals Fused-silica capillaries (75 ␮m I.D. × 375 ␮m O.D., 150 ␮m I.D. × 375 ␮m O.D.) were obtained from Yongnian Optical Fiber Factory (Hebei, China). Acrylamide (99+%), N-acryloxysuccinimide (99%), benzamidine hydrochloride hydrate (98%), N,N-methylenebisacrylamide (99%), N,N,N ,N tetramethylethylenediamine (TEMED), ammonium persulfate, poly(ethylene glycol) (PEG, Mw 10,000) and ␥-methacryloxypropyl trimethoxysilane (␥-MAPS, 98%) were ordered from Acros (New Jersey, USA). Ethylene dimethacrylate

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2.3. Preparation of monolithic capillary enzymatic reactor

(97+%), cyclohexanol (99+%), 1-dodecanol (99.5+%), and N␣-benzoyl-l-arginine ethyl ester (BAEE) were purchased from Fluka (Buchs, Switzerland). Azobisisobutyronitrile (AIBN) was purified by recrystallization from methanol. Trypsin (bovine pancreas), cytochrome c (horse heart), ovalbumin (chicken), lysozyme (chicken egg) and insulin (bovine) were obtained from Sigma (St. Louis, MO, USA). Formic acid (88%), acetic acid, urea, dimethyl sulfoxide (DMSO) and ammonium bicarbonate (NH4 HCO3 ) were from Beijing Chemical Reagent Company (Beijing, China). Acetonitrile was purchased from Scharlau Chemie (Barcelona, Spain). Water was purified by a Milli-Q system (Millipore, Molsheim, France). All inorganic reagents were analytical-reagent grade, and all solvents were HPLC grade.

The capillaries were first rinsed respectively by methanol, water, 0.1 mol/L HCl, water, 0.1 mol/L sodium hydroxide and methanol. Then, they were dried in a stream of nitrogen at 70 ◦ C. After that, a 50% solution of ␥-MAPS in methanol was pumped through the capillaries overnight at room temperature, followed by washing with methanol and drying with nitrogen gas. With such a procedure, the monolithic supports could be covalently anchored to the inner wall of capillaries. The polymerization mixture consists of acrylamide, NAS and EDMA as monomers, cyclohexanol and 1-dodecanol as porogenic solvent, and AIBN as thermo-initiator. Acrylamide was used as the inert hydrophilic comonomer to adjust the hydrophilicity of the monolithic supports, and NAS was the active monomer, offering extremely high activity to react with the amino group of proteins. In the reaction, monomers were polymerized with crosslinker to build the skeleton of the polymer, making the succinimide functional radical distribute along the monolithic column. The mixture of dodecanol and cyclohexanol was chosen as the porogenic reagents. It was confirmed that narrow pore distribution and large pore size of monolithic support could be achieved from the thermo-initiated polymerization in such a porogenic system at 55 ◦ C [42], and the amount of dodecanol contained in the polymerization mixture only had minor effect on the pore volume [43]. Therefore, the percentage of dodecanol in the dual porogenic solvent was chosen as 15% (w/w) without further optimization. The compositions of the individual polymerization mixtures were listed in Table 1. The polymerization mixture was heated to 60 ◦ C with vortex shaking and sonicating for completely dissolution prior to the addition of thermo-initiator. After mixing, AIBN was dissolved into the mixture by sonication. Later, the polymerization solution was purged with nitrogen for 15 min. Subsequently, the solution was introduced to the pre-treated capillaries by a syringe to the desired length. With both ends sealed by silicon rubbers, the capillaries were heated at 55 ◦ C for 6 h. After the polymerization was completed, methanol was pumped through the monoliths to wash out the porogenic solvents and other compounds. Before usage, each column was inspected under the microscope to check the uniformity of the stationary phase, and further investigated by SEM. Then, trypsin was coupled to the support by continuously introducing 2.5 mg/mL trypsin in a buffer (pH 8.0) containing

2.2. Equipment Capillary electrophoresis (CE) experiments were performed on a Beckman Coulter P/ACE MDQ system (Beckman, Fullerton, CA, USA). A length of 31.5 cm fused-silica capillary was used for enzyme activity study. Nano-HPLC experiments were carried out on TriSep2010GV capillary electrochromatography system (Unimicro Technologies, Inc. Pleasanton, CA). Finnigan LCQDUO ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) was used for ESI-MS detection. Total ion chromatograms and mass spectra were recorded on a personal computer with the Xcalibur software version 1.4. Mass calibration and tuning were performed in the positive ion mode by direct infusion of a solution of caffeine (Sigma), methionyl-arginyl-phenylalanyl-alanine (Finnigan) and Ultramark 1621 (Finnigan). The tandem mass spectra analyzing and protein database searching were operated with the Bioworks software version 3.1. The Nano-LC-MS/MS system was established by using a length of 17 cm 75 ␮m I.D. capillary with tapered end and 3 ␮m size of reversed phase silica as the separation column to directly couple with mass spectrometer without extra spray emitter connected. A high voltage was applied at the inlet of the column to conduct the electrospray potential. The tip of the separation capillary was adjusted just in front of the orifice of the heated capillary in mass spectrometer. Scanning electron microscope (SEM) images were collected using a JEOL JSM-6360LV scanning electron microscope (JEOL, Tokyo, Japan).

Table 1 Composition of polymerization mixture Column

A B C D E

Monomers (wt.%)

Porogen (wt.%)

Monomer: porogen (wt.%)

EDMA

NAS

AA

Cyclohexanol

1-Dodecanol

50 50 50 50 50

20 20 20 20 20

30 30 30 30 30

85 85 85 85 85

15 15 15 15 15

15:85 20:80 25:75 30:70 40:60

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0.5 M sodium chloride, 0.2 M sodium hydrogen carbonate and 50 mM benzamidine for 1 h at 25 ◦ C. The monolithic trypsin microreactor prepared in aqueous buffer could be simply described as follow [30]. The polymerization solution was prepared by dissolving 20 mg of acrylamide, 30 mg of N,N-methylenebisacrylamide, and 30 mg of PEG in 1 mL of 0.2 M sodium bicarbonate and 0.5 M sodium chloride buffer (pH 8.0). After that, 500 ␮L of this solution were taken out and 2 ␮L of 20% (v/v) TEMED were added. A 5 ␮L DMSO solution of N-acryloxysuccinimide with concentration of 140 mg/mL was then added. After sonicated for 1 min, 2 ␮L of 20% (w/v) ammonium persulfate (APS) was added to initiate polymerization. After 30 s, a 19-␮L aliquot of this solution was removed and quickly mixed with 1 ␮L of freshly prepared trypsin (20 mg/mL in a buffer containing 0.5 M benzamidine). The mixture was filled into the activated capillaries, and the polymerization was carried out at room temperature for 1 h with both ends sealed. This column was named as column F. After polymerization and enzyme immobilization, all enzymatic reactors were rinsed and filled with 0.5 M sodium chloride and 0.2 M sodium hydrogen carbonate buffer (pH 8.0) containing 10 mM CaCl2 and 0.02% NaN3 , and stored in the freezer at 4 ◦ C before usage. 2.4. Assay of enzymatic activity Activity of the immobilized enzyme was determined by the hydrolysis of 20 mM BAEE at various volumetric flow rates at 25 ◦ C. The values of Michaelis constant (Km ) and maximum velocity (Vmax ) were obtained by the analysis of initial velocities at a series of BAEE concentration when the substrate was pumped through the microreactor at a constant volumetric flow rate at 25 ◦ C. The generated products were amalyzed by CE using a 50 mM Tris–HCl (pH 7.5) buffer. The reacted BAEE was determined by measuring the peak area of the substrate. 2.5. Protein digestion Protein samples were first denatured in 50 mM NH4 Ac/ NH3 H2 O buffer containing 8 M urea for 1 h at 37 ◦ C, followed by dilution with 50 mM NH4 Ac/NH3 H2 O (pH 8.0) buffer to the concentration of urea below 1 M. The in-solution digestion was performed by adding trypsin into the protein solution at a substrate-to-enzyme ratio of 50:1, and the solution was incubated at 37 ◦ C for 12 h. After digestion, 1 ␮L formic acid was added into the solution to stop the reaction. The off-line protein digestions using immobilized reactor were carried out by pumping sample solution through the enzymic reactor at a constant flow rate. Products were collected, separated and detected using nano-LC-MS/MS. The on-line digestion system was illustrated in Fig. 1. Protein solution was pushed through the microreactor using a syringe pump and directly transferred into a nano-scale 4-port injection valve at a constant flow rate before nano-LC separation and MS detection.

Fig. 1. Schematic diagram of the on-line digestion coupled with nano-LCMS/MS system.

2.6. Preparation of the separation column The separation columns were prepared from fused-silica capillaries (75 ␮m I.D. × 375 ␮m O.D.) packed with 3 ␮m C-18 silica (Great Eur-Asia Sci&Development Co. Ltd., Beijing, China). One end of the capillary was treated by applying heat from a gas jet while pulling gently. The resulting tapered tip was later trimmed to the desired tip inside diameter (∼20 ␮m) using a cleaving stone under microscope observation. The tapered end of the capillary would be served as the outlet frit of the separation column. The slurry of acetone and the packing material was prepared at a ratio of 10:1 (v/w) and packed at a pressure of 30 MPa to a certain length. The inlet frit was sintered by heating the outer wall of the capillary through a certain length of resistance coil. 2.7. Protein identification using SEQUEST search program Protein identification was performed using the BioWorks software with SEQUEST search program. The MS/MS raw data obtained from the LC-MS/MS analysis were used for database search. Trypsin was selected as protein cleavage specificity. Both b-ions and y-ions were included in the database search. Database searching of cytochrome c digests was performed in horse.fasta database. The protein mixture was run against the nr.fasta (nonredundant) database. All databases were downloaded from free ftp website of the National Centre for Biotechnology Information (ftp://ftp.ncbi.nih.gov, Bethesda, MD, USA). The Xcorr (cross-correlation) and DeltaCn values were used as a primary indicator for identifying proteins. 3. Results and discussion 3.1. Characteristics of the monolithic microreactor The effects of the monomers to porogenic solvent ratio in the polymerization solution were investigated. A series of monolithic columns were prepared from the polymerization mixture listed in Table 1. Monoliths in columns A and B were pushed out from the capillaries when methanol was pumped through them with a pressure of 10 MPa applied, because of their relative looser structure. After using a low pressure methanol rinse as alternate, a discrete and fragmented structure of monolith along columns A and B was found under microscope observation. It is because that the amounts of crosslinker in the monoliths A and

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Fig. 2. SEM micrographs of internal structure of monoliths C, D and E magnified by 3000 times (from left to right).

B were too low to make the network formation constructed, and resulting in poor covalent bridge with the capillary inner wall as well. In contrast, there was no obvious breakage of the structure in monoliths C, D and E along the columns after 10 MPa pressure applied. Fig. 2 showed the SEM graphs of the internal morphologies of monolithic columns C, D and E with magnification of 3000×, respectively. The uniform and small sizes of the particles could provide a high surface area for the immobilization of enzyme, and the macroporous structure resulted in low backpressure and high throughput of samples. As the SEM images showed, there was little difference between the micro structure of monoliths C and D, however, the particle size of monolith E was obviously larger than that of the former ones. It may be ascribed to the high content of monomers in monolith E. The effect of pressure on the flow rate for monoliths C, D and E with enzyme immobilized was studied by using a micro flow rate pump operated under constant pressure mode and weighting the effluent passed through the monolithic column. Fig. 3 showed that good linear relationships between the flow rate and the applied pressure were achieved on monoliths C, D and E with a length of 6 cm in the pressure range of 0.5–2.5 MPa, respectively. The results showed that all three columns had high throughput capabilities, among which the flow rate of column C could reach 2.2 ␮L/min when only 2.5 MPa pressure was applied. The results were as good as that of the published work [23]. So, columns C, D and E were selected as the supports for enzyme immobilizing in our work.

Fig. 3. Effect of back pressure on flow rate for monoliths C, D and E with immobilized trypsin. Experimental conditions: mobile phase, 50 mM Tris buffer.

The activities of monoliths C, D and E immobilized with trypsin were compared using BAEE as the substrate. A 20 mM BAEE solution was pumped through microreactors with a length of 2 cm at the same flow rate of 500 nL/min. Then the products were collected and separated by CE with UV detection, which were shown in Fig. 4. Although all three microreactors showed hydrolysis activities towards BAEE, column C showed the highest activity, while column E offered the lowest, which could be explained that with the percentage of monomer in polymerization solution increasing, the size of particle in the monolith became larger and the specific surface area was decreased. Therefore, the amounts of enzyme immobilized on the supports were decreasing, resulting in less enzymatic activity. According to above results, monolith C was chosen as the optimal microreactor material for the following application. 3.2. Activities of enzymatic microreactors The activity of an enzyme, and consequently that of the microreactor, was characterized by the values of the Michaelis constant (Km ) and maximum velocity (Vmax ), which could be obtained from the rewritten form of the Michaelis–Menten equation [44]. The most commonly used transformation is the doublereciprocal or Lineweaver–Burke plot. The Lineweaver–Burk equation is 
1 1 Km 1 = , + v0 Vmax [S] Vmax

Fig. 4. Electropherograms of products after BAEE pumped through monoliths C, D and E with immobilized trypsin. Experimental conditions: 20 mM BAEE (50 mM Tris–HCl, pH 8.0); 50 mM Tris–HCl (pH 7.5), 15 kV, 0.5 psi 10 s, 25 ◦ C; flow rate: 500 nL/min, 3 cm of microreactor each.

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Fig. 5. Lineweaver–Burke plots for trypsin immobilized on monoliths C and F. Experimental conditions: 1 cm microreactor (150 ␮m I.D.), 300 nL/min, 25 ◦ C, 10–100 mM BAEE in 50 mM Tris–HCl (pH 8.0).

in which the value of 1/v0 are plotted against 1/[S] [44–46]. Although double-reciprocal plot, in which high measuring errors are existed as low concentration substrates used, is not the most accurate method for determining kinetic constants, it is easily obtained and understood. In this paper, the concentrations of BAEE were selected in the range of 10–100 mM for minimize the errors of the experiment. The values of Km and Vmax of trypsin immobilized on monoliths C and F were obtained through analyzing the initial velocities of substrate with a series of concentrations in the same microreactor at a flow rate of 300 nL/min. Fig. 5 showed the plots for the conversion of BAEE in enzymatic microreactors prepared by immobilization of trypsin on monoliths C and F. The values of Km were 68.1 and 49.7 mM and the values of Vmax were 41.5 ␮mol/min and 32.2 ␮mol/min for columns C and F, respectively. The higher Km value means the weaker affinity between substrate and enzyme immobilized on monolith. It revealed that the aqueous prepared supports have the better hydrophilicity than the alcohol polymerized matrix, so the substrate was prone to the enzyme coupled on monolith F resulted in a lower value of Km . The higher Vmax value was obtained on column C because that the more NAS was composed in the skeleton of the monolithic matrix. However, the value of Km not only measures the affinity between enzyme and substrate but also reflects the concentration of substrate required when initial velocity is half of its maximum value. Thus, although the maximum velocity is higher, a higher concentration of substrate must be used to reach this velocity. Fig. 6 shows the effect of BAEE flow rate on the enzymatic activity of trypsin immobilized on monoliths C and F. As expected, the enzymatic activity increases with the flow rate, which is presumably attributed to the increase of the mass transfer of the substrate towards the fixed enzyme molecules and the reduce of the stagnant diffusive solvent layer at the monolithic materials. Monoliths C and F with NAS and acrylatemide as monomers have the good hydrophilicity, therefore, the good accessibility to enzyme. However, the configuration of monolith F is restricted

Fig. 6. Effect of flow rate of the substrate solution on the activity of trypsin immobilized on monoliths C and F. Experimental conditions: 1 cm microreactor (150 ␮m I.D.), 20 mM BAEE in 50 mM Tris–HCl (pH 8.0), 25 ◦ C, flow rates: 200 nL/min–1 ␮L/min.

by the poor solubility of monomers in aqueous buffer so that only low content of monomer and crosslinker could be used. Furthermore, the monolith such as F, offers soft, loose and gel-like structure, resulting in a considerable shrinking after the buffer is volatilized out of the matrices. In addition, the pressure resistant capability is poor. The activity of enzyme bounded on monolith F increases slowly with the flow rate, and shows a plat shape at the higher flow rate range as shown in Fig. 6. As the flow rate increases further, the inlet part of the monolith is easy to break down, even the whole monolithic bed could be pushed out of the capillary. In contrast, the monoliths polymerized in alcohol porogenic solvent system are rigid enough to support a backpressure of 20 MPa, and the activity of the immobilized trypsin increases rapidly with the flow rate in our experimental range. By comparison, in low flow rate range, the activity of trypsin on monolith F is higher than that in C, because of the more loose and hydrophilic skeleton structure compared with that of C. At high flow rate, trypsin is more active in monolith C which is resulted from the more efficient mass transport. Therefore, for high throughput digestion, monolith C polymerized in alcohol porogenic solvent system is the optimal choice. 3.3. Comparison between in-solution digestion and on-column digestion by nano-LC-MS/MS system The hydrolysis reaction of macromolecule, such as protein digestion, is more difficult than that for the small one. As a short length of reactor (2 or 4 cm) was used, the flowrates applied for protein digestion using immobilized reactor should be reduced for ensuring enough resident time of protein to complete the reaction. In our study, a low flowrates of 300 nL/min was applied for protein off line digestion. The comparison of the traditional in-solution digestion to the on-column reaction is investigated by nano-LC-MS/MS system using cytochrome c as the sample. Fig. 7 shows the base peak chromatograms of peptides produced from the two digestion protocols mentioned above. The similar profiles of

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Fig. 7. Base peak chromatograms for products of cytochrome c digested in solution and through microreactor. Experimental conditions: 0.1 mg/mL cytochrome c (50 mM NH4 Ac/NH3 H2 O, pH 8.0), 37 ◦ C. In solution: enzyme:protein = 1:50, 12 h; microreactor: 2 cm (150 ␮m I.D.), flow rate 300 nL/min. Nano-HPLC-MS/MS conditions: column: 75 ␮m × 17 cm, 3 ␮m C-18; HPLC conditions: mobile phase: (A) 95% ACN (containing 0.1% formic acid), (B) 5% ACN (containing 0.1% formic acid); flow rate: 50 ␮L/min, backpressure: 2000 psi; gradient: 0 min, 5% A; 25 min, 40% A; 45 min, 80% A; 46 min, 100% A; 50 min, 100%; voltage: 2 kV; MS condition: heated capillary temperature:150 ◦ C, collision energy: 35%.

chromatogram are achieved. Most of chromatographic peaks are detected and identified by MS/MS, and the corresponding database researching results are listed in Table 2. The magnitude of the cross-correlation value indicates the quality of the match between the sequence and the spectrum, and the difference between the normalized cross-correlation score to the second ranked sequence shows the quality of the match versus all the other top ranking sequences in the

database. The thresholds of Xcorr ≥ 1.5, DeltaCn ≥ 0.085 and Percent Ions ≥ 30% are used for unambiguous identification of cytochrome c, yielding the sequence coverage of 58.65 and 59.62% for on-column and in-solution digestion, respectively. Although the similar peptide fragments and sequence coverage were achieved, the residence time of protein in microreactor is only 27 s, more than one thousand time faster than the in-solution digestion, which takes about 12 h.

Table 2 Results of the database searchinga from the nano-LC-MS/MS analysisb of peptides digested in solution and through monolithic microreactorc

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11

Sequence

Position

Peptide mass [M + H]+ (Da)

Charge

K.TGPNLHGLFGR.K R.KTGQAPGFTYTDANK.N K.TGQAPGFTYTDANK.N K.TGQAPGFTYTDANKNK.G K.GITWKEETLMEYLENPKK.Y K.EETLMEYLENPK.K K.EETLMEYLENPKK.Y K.KYIPGTK.M K.MIFAGIK.K K.MIFAGIKK.K R.EDLIAYLK.K

28–38 39–53 40–53 40–55 56–73 61–72 61–73 73–79 80–86 80–87 92–99

1169.32 1599.73 1471.55 1713.83 2210.54 1496.67 1624.84 806.97 780.01 908.19 965.13

2 2 2 2 3 2 2 2 1 2 1

Digestion time Sequence coverage (%)

On-column digestion √ √ √ √ √ √ √ √

In-solution digestion √ √ √ √ √

√

√ √

27 s 58.65

12 h 59.62

Thresholds: Xcorr ≥ 1.5, DeltaCn ≥ 0.085 and Percent Ions ≥ 30%. Database: horse.fasta. Nano-HPLC-MS/MS conditions: column: 75 ␮m × 17 cm, 3 ␮m C-18; HPLC conditions: mobile phase: A, 95% ACN (containing 0.1% formic acid); B, 5% ACN (containing 0.1% formic acid); flow rate: 50 ␮L/min, backpressure: 2000 psi, gradient: 0 min, 5% A; 25 min, 40% A; 45 min, 80% A; 46 min, 100% A; 50 min, 100%. Voltage: 2 kV; MS condition: heated capillary temperature: 150 ◦ C; mass rang: 400–2000; collision energy: 35%. c Conditions: in-solution digestion: 100 ug/mL cytochrome c, enzyme:protein = 1:50, 37 ◦ C; on-column digestion: 100 ug/mL cytochrome c, flow rate: 300 nL/min, 37 ◦ C. a

b

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Fig. 8. Base peak chromatograms for the digests of 0.1 and 1 mg/mL cytochrome c microreactor digestion, together with that for the unreacted protein. Experimental conditions: Cytochrome c (50 mM NH4 Ac/NH3 H2 O, pH 8.0), 37 ◦ C, flow rate 300 nL/min. Nano-HPLC-MS conditions as Fig. 7.

3.4. Effect of sample concentration The on-column digestion is also affected by the concentration of proteins. The comparison between the digestion of 0.1 and 1 mg/mL cytochrome c solution by microreactor is studied with nano-LC-MS/MS. Fig. 8 shows the base peak chromatogram of the produced peptides, from which it could seen that it is difficult to achieve complete conversion for high concentration protein since the trypsin sites immobilized on the monolithic microreactor could be saturated. Therefore, such a enzymatic reactor has shown promising in the high throughput digestion of low abundance proteins. The conversion of digestion would be more efficient and complete as low concentration proteins used as substrates. However, the velocity of reaction would be decreasing with the reduction of protein concentration. As a promising choice, the concentration of protein in ug/mL level would be preferred in on-column digestion. Unfortunately, in our study, the concentration of protein was restricted by the scale of the injection valve and the dead volume in connection, a pg/uL protein solution should be supplied to ensure enough sensitivity for MS/MS detection. 3.5. On-line protein digestion, separation and identification system 3.5.1. The influence of the flow rate on the microreactor performance A very simple on-line system is established for the fast digestion, separation and identification of proteins. In our design, the denatured protein solution is pushed through a length of 2 or 4 cm microreactor using a syringe pump and directly transferred into a nano-scale 4-port injection valve before entering the nanoLC–MS/MS system.

The effect of flow rate on the on-column digestion is investigated with a microreactor (length: 2 cm, I.D.: 100 ␮m). Fig. 9 shows the base peak chromatogram of on-line digestion products of 0.2 mg/mL cytochrome at the flow rate of 50, 200 and 500 nL/min, respectively. Two cytochrome c fragments ([M + H]+ 678.8 and 780.0) are chosen for the relative intensity comparison. As shown in Table 3, the MS signals of both peptides are reduced with the increase of the flow rate, which means that the higher the digestion flow rate, the less fragments produced. Correspondingly, the number of identified peptides and sequence coverage are reduced. When the flow rate of protein past through the microreactor is up to 1 ␮L/min, with the corresponding residence time of 7 s in the microreactor, just five peptides are identified. Even though, a sequence coverage of 54.81% could be achieved in such a short digestion time. 3.5.2. Identification of four standard proteins mixture A mixture of four standard proteins, including insulin (Mw 5733), lysozyme (Mw 13,930), cytochrome c (Mw 12,360) and ovalbumin (Mw 44,000), was used to test the performance of the on-line system. The total amount of 8 ng protein (2 ng for each protein) is transferred through a length of 4 cm microreactor into the nano-scale injection valve at 100 nL/min with the residence time of 142 s. Then, the digests are directly separated and detected using Nano-LC-MS/MS. All four proteins are identified after database searching using multiple thresholds of Xcorr ≥ 1.5, DeltaCn ≥ 0.085 and Percent Ions ≥ 30%. The identified peptides and protein sequence coverage are listed in Table 4. Cytochrome c is unambiguously identified with the highest score and eight peptides are found in the database, yielding a sequence coverage of 57.69%, higher than that for ovalbumin, lysozyme and insulin respec-

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173

Fig. 9. Base peak chromatograms for the digests of cytochrome c at different flow rates. Experimental conditions: 0.2 mg/mL cytochrome c (50 mM NH4 Ac/NH3 H2 O, pH 8.0), 37 ◦ C, flow rate 50, 200 and 500 nL/min. Nano-HPLC-MS conditions as Fig. 7. Table 3 The results of protein digestion and identification using on-line system at various flow rates Flow rate (nL/min)

50 200 500

Residence time (s)

142 36 14

Number of identified peptides

Sequence coverage (%)

Intensity of selective peaks m/z 678.8

m/z 780.0

14 11 8

72.12 71.15 54.81

5.37E + 07 6.99E + 06 3.63E + 06

4.88E + 06 2.36E + 06 9.92E + 05

Experimental conditions: sample: 0.2 mg/mL cytochrome c; on-line system conditions: microreactor: 100 ␮m I.D., 375 ␮m O.D., 2 cm; injection volume: 10 nL; flow rates: 50, 200, 500 nL/min; nano-HPLC-MS conditions were the same as Table 2. Table 4 The database searching results of digestion of protein mixture Protein

Sequence

Cytochrome c

TGPNLHGLFGR TGQAPGFTYTDANK TGQAPGFTYTDANKNK EETLMEYLENPKK MIFAGIKK KTEREDLIAYLK TEREDLIAYLK EDLIAYLK

Ovalbumin Lysozyme Insulin

ELINSWVESQTNGIIR FESNFNTQATNR GFFYTPKA

Peptide mass [M + H] (Da)

Charge

Xcorr

Sequence coverage (%)

28–38 40–53 40–55 61–73 80–87 88–99 89–99 92–99

1169.32 1471.55 1713.83 1624.84 908.19 1479.7 1351.53 965.13

2 2 2 3 2 2 1 2

3.01 4.28 4.66 3.78 2.54 3.11 2.97 1.65

57.69

143–158 34–45 44–51

1860.06 1429.48 931.07

2 2 2

4.14 3.26 1.96

4.16 9.30 15.69

Position

Experimental conditions: 8 ng proteins mixture (containing cytochrome c, ovalbumin, lysozyme and insulin 2 ng each) in 50 mM NH4 Ac/NH3 H2 O, pH 8.0), 37 ◦ C; 4 cm microreactor (100 ␮m I.D.), flow rate: 100 nL/min; nano-HPLC-MS conditions gradient: 0 min, 5% A; 30 min, 40% A; 60 min, 80% A; 61 min, 100% A; 65 min, 100%; other condition as Table 2 database searching thresholds: Xcorr ≥ 1.5, DeltaCn ≥ 0.085 and Percent Ions ≥ 30%. Database: nr.fasta.

tively. In addition, the Xcorr values for all digested peptides from four proteins are high enough to identify the proteins with high confidence. The result means that the proteins could be identified through our on-line digestion system with even in ng level of the mixture of proteins with different molecular weights.

4. Conclusions A novel monolithic tryptic microreactor with high proteolytic activity was prepared in alcohol porogenic system, and the reduced immobilization time was achieved by using NAS as monomer. The very short digestion time was achieved with

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on-column reaction compared with the longer reaction time required to achieve similar sequence coverage using traditional in-solution approaches. A simple on-line protein digestion and identification system was developed by using microreactor coupled with a nanoLC-MS system and evaluated with a mixture of proteins. The very rapid protein digestion and identification with high confidence confirmed the potential application of this system in proteomics research. Additionally, a statement should be stressed that the volume of sample injected in the nano-LC was restricted by the 10-nL injection loop in our on-line system, so a high concentration of sample, in high pg/␮L level, should be used to supply enough amount of peptide for MS/MS detection with enough sensitivity. Our further study will focus on the low-concentration proteins digestion with new concentration methods to increase the sample injection. Acknowledgements Authors are grateful for the financial support from National Program on Key Basic Research Projects (973 Program) of China (Nos. 2001CB51020 and 2004CB520804), Natural Nature Science Fundation (Nos. 20435020 and 20405014), Science and Technology Foundation of Liaoning Province and “100 Talent” Project from Chinese Academy of Sciences. References [1] J. Godovac-Zimmermann, L.R. Brown, Mass Spectrom. Rev. 20 (2001) 1. [2] R. Aebersold, D.R. Goodlett, Chem. Rev. 101 (2001) 269. [3] R.J. Deshaies, J.H. Seol, W.H. McDonald, G. Cope, S. Lyapina, A. Shevchenko, A. Shevchenko, R. Verma, J.R. Yates III, Mol. Cell. Proteomics 1 (2002) 3. [4] S.P. Gygi, R. Aebersold, Curr. Opin. Chem. Biol. 4 (2000) 489. [5] J.R. Yates III, J. Mass Spectrom. 33 (1998) 1. [6] Q. Xia (Ed.), Research Technologies and Advances in Protein Chemistry, Science Press, Beijing, China, 1997, pp. 16–17, pp. 240–244. [7] I.M. Lazar, R.S. Ramsey, J.M. Ramsey, Anal. Chem. 73 (2001) 1733. [8] J.P. Chang, D.E. Kiehl, A. Kennington, Rapid Commun. Mass Spectrom. 11 (1997) 1266. [9] G.W. Slysz, D.C. Schriemer, Anal. Chem. 77 (2005) 1572. [10] G.T. Hermanson, A.K. Mallia, P.K. Smith (Eds.), Immobilizied Affinity Ligand Techniques (Chinese Translation Edition), Science Press, Beijing, 1996, pp. 29–34, pp. 125–128. [11] M. Bengtsson, S. Ekstr¨om, G. Marko-Varga, T. Laurell, Talanta 56 (2002) 341. [12] B.E. Slentz, N.A. Penner, F.E. Regnier, J. Chromatogr. A 984 (2003) 97. [13] G.W. Slysz, D.C. Schriemer, Rapid Commun. Mass Spectrom. 17 (2003) 1044. [14] E. Bonneil, M. Mercier, K.C. Waldron, Anal. Chim. Acta 404 (2000) 29.

[15] L.J. Jin, J. Ferrance, J.C. Sanders, J.P. Landers, Lab. Chip 3 (2003) 11. [16] K. Yamada, T. Nakasone, R. Nagano, M. Hirata, J. Appl. Polym. Sci. 89 (2003) 3574. [17] K. Sakai-Kato, M. Kato, T. Toyo’oka, Anal. Chem. 74 (2002) 2943. [18] K. Sakai-Kato, M. Kato, T. Toyo’oka, Anal. Chem. 75 (2003) 388. [19] J. Gao, J. Xu, L.E. Locascio, C.S. Lee, Anal. Chem. 73 (2001) 2648. [20] J.W. Cooper, J. Chen, Y. Li, C.S. Lee, Anal. Chem. 75 (2003) 1067. [21] M. Petro, F. Svec, J.M.J. Fr´echet, Biotechnol. Bioeng. 49 (1996) 355. [22] S. Xie, F. Svec, J.M.J. Fr´echet, Biotechnol. Bioeng. 62 (1999) 30. [23] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Fr´echet, Anal. Chem. 74 (2002) 4081. [24] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Fr´echet, J. Proteome Res. 1 (2002) 563. [25] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Fr´echet, Anal. Chem. 75 (2003) 5328. [26] E. Calleri, C. Temporini, E. Perani, C. Stella, S. Rudaz, D. Lubda, G. Mellerio, J.-L. Veuthey, G. Caccialanza, G. Massolini, J. Chromatogr. A 1045 (2004) 99. [27] E. Calleri, C. Temporini, E. Perani, A.D. Palma, D. Lubda, G. Mellerio, A. Sala, M. Galliano, G. Caccialanza, G. Massolini, J. Proteome Res. 4 (2005) 481. [28] F. Svec, J.M.J. Fr´echet, Science 273 (1996) 205. [29] D. Josic, A. Buchacher, A. Jungbauer, J. Chromatogr. B 752 (2001) 191. [30] A.K. Palm, M.V. Novotny, Rapid Commun. Mass Spectrom. 18 (2004) 1374. [31] M. Ye, S. Hu, R.M. Schoenherr, N.J. Dovichi, Electrophoresis 25 (2004) 1319. [32] E.F. Hilder, F. Svec, J.M.J. Fr´echet, Electrophoresis 23 (2002) 3934. [33] Y. Ishihama, J. Chromatogr. A 1067 (2004) 73. [34] Y. Shen, R. Zhao, S.J. Berger, G.A. Anderson, N. Rodriguez, R.D. Smith, Anal. Chem. 74 (2002) 4235. [35] C. Ihling, K. Berger, M.M. H¨ofliger, D. F¨uhrer, A.G. Beck-Sickinger, A. Sinz, Rapid Commun. Mass Spectrom. 17 (2003) 1240. [36] B. Devreese, K.P.C. Janssen, F. Vanrobaeys, F. Van Herp, G.J.M. Martens, J. Van Beeumen, J. Chromatogr. A 976 (2002) 113. [37] M. Mazereeuw, A.J.P. Hofte, U.R. Tjaden, J. van der Greef, Rapid Commun. Mass Spectrom. 11 (1997) 981. [38] J.C. Hannis, D.C. Muddiman, Rapid Commun. Mass Spectrom. 12 (1998) 443. [39] A.J. Link, J. Eng, D.M. Schieltz, E. Carmack, G.J. Mize, D.R. Morris, B.M. Garvik, J.R. Yates III, Nat. Biotechnol. 17 (1999) 676. [40] D.A. Wolters, M.P. Washburn, J.R. Yates III, Anal. Chem. 73 (2001) 5683. [41] M.P. Washburn, D. Wolters, J.R. Yates III, Nat. Biotechnol. 19 (2001) 242. [42] C. Viklund, F. Svec, J.M.J. Fr´echet, Chem. Mater. 8 (1996) 744. [43] T. Rohr, C. Yu, M.H. Davey, F. Svec, J.M.J. Fr´echet, Electrophoresis 22 (2001) 3959. [44] H.R. Horton, L.A. Moran, R.S. Ochs, J.D. Rawn, K.G. Scrimgeour, Principles of Biochemistry, English Reprint, third ed., Science Press and Pearson Education North Asia Limited, Beijing, China, 2002, pp. 135–141. [45] S. Ekstrom, P. Onnerfjord, J. Nilsson, M. Bengtsson, T. Laurell, G. Marko-Varga, Anal. Chem. 72 (2000) 286. [46] A.G. Hadd, D.E. Raymond, J.W. Halliwell, S.C. Jacobson, J.M. Ramsey, Anal. Chem. 69 (1997) 3407.