Ion-exchange-membrane-based enzyme micro-reactor coupled online with liquid chromatography–mass spectrometry for protein analysis
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- Zhou, Z., Yang, Y., Zhang, J. et al. Anal Bioanal Chem (2012) 403: 239. doi:10.1007/s00216-012-5812-2
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In this article, we developed a membrane-based enzyme micro-reactor by directly using commercial polystyrene–divinylbenzene cation–exchange membrane as the support for trypsin immobilization via electrostatic and hydrophobic interactions and successfully applied it for protein digestion. The construction of the reactor can be simply achieved by continuously pumping trypsin solution through the reactor for only 2 min, which was much faster than the other enzyme immobilization methods. In addition, the membrane reactor could be rapidly regenerated within 35 min, resulting in a “new” reactor for the digestion of every protein sample, completely eliminating the cross-interference of different protein samples. The amount and the activity of immobilized trypsin were measured, and the repeatability of the reactor was tested, with an RSD of 3.2% for the sequence coverage of cytochrome c in ten digestion replicates. An integrated platform for protein analysis, including online protein digestion and peptide separation and detection, was established by coupling the membrane enzyme reactor with liquid chromatography–quadrupole time-of-flight mass spectrometry. The performance of the platform was evaluated using cytochrome c, myoglobin, and bovine serum albumin, showing that even in the short digestion time of several seconds the obtained sequence coverages was comparable to or higher than that with in-solution digestion. The system was also successfully used for the analysis of proteins from yeast cell lysate.
KeywordsIon exchange membraneEnzyme micro-reactorProtein analysisLiquid chromatographyMass spectrometryLC-MS
“Shotgun” has become a well-accepted strategy for proteomic research, which typically involves protein digestion and peptide separation and detection by liquid chromatography–mass spectrometry (LC-MS). High-throughput analysis of proteins could not be realized without rapid, efficient, and reliable methods for sample preparation. However, traditional in-solution digestion has been a bottleneck for the development of proteomic research, due to its several drawbacks including being time consuming and autolysis of enzyme. The enzyme micro-reactor, as a good alternative to in-solution digestion, combines the advantages of both enzyme immobilization and micro-reactors, such as high localized enzyme concentration with little autolysis, fast mass transfer, little sample consumption, and so on.
Typically in enzyme micro-reactors, the enzyme is immobilized on a kind of supporting material, like packed beads, monoliths [1–9], membranes [10–14], and the inner-walls of capillary columns or microchip channels  through physical adsorption, sol–gel entrapment, covalent binding, or electrostatic interactions. Among these carriers, the capillary monolithic column has been widely utilized due to its low back pressure and excellent mass transfer property, in which the enzyme is usually immobilized via covalently binding to the functional monomers like glycidyl methacrylate [1, 3–5, 8, 9], N-acryloxysuccinimide [2, 6, 7], etc. Besides the self-synthesized monolithic columns, many commercial membranes with large surface to volume ratios could be directly used to carry enzymes, such as poly(vinylidene fluoride) (PVDF) film, nylon membrane, and cellulose film. For example, a microfluidic system using PVDF membrane to immobilize trypsin via hydrophobic interaction was conducted by Lee and coworker for protein digestion , and the membrane reactor was further coupled with membrane chromatography to achieve online peptide separation . A miniaturized reactor was also presented by placing a small piece of PVDF membrane inside a capillary fitting, and it could perform rapid proteolytic digestion in seconds for a protein of less than 10−8 M, consuming very little amount of sample [10, 13]. Moreover, to prevent the leakage of enzyme from the membranes, some more stable methods for enzyme immobilization have been reported, such as avidin–biotin molecular recognition , chemical binding to modified poly(methyl methacrylate) composite membrane , etc.
Among various immobilization methods, electrostatic interaction should be preferential since it is stronger than physical adsorption, avoiding the enzyme leakage. Although covalent binding is much more stable, multiple steps of chemical reactions are usually required to complete the immobilization, which might lead to the loss of enzyme activity to some extent. In contrast, the immobilization procedure through electrostatic interaction is simple, rapid, and mild. Immobilized capillary enzyme reactors created by ionic binding technology have been reported for rapid screening of enzyme inhibitors. The enzymes were simply immobilized on the inner wall of fused silica capillary coated with cationic polyelectrolyte by pumping the enzyme solution through the capillary using a capillary electrophoresis instrument, and the reactor could be easily renewed by flushing with 1 M NaCl [18, 19]. Xu and coworkers  have recently presented a membrane reactor using poly(styrene sulfonate)-modified nylon membrane as the enzyme support, on which trypsin was adsorbed via electrostatic interaction. The sequence coverage of 84% for bovine serum albumin (BSA) was obtained, and the reactor showed high tolerance to surfactants, e.g., sodium dodecyl sulfate, which were used for protein denaturation. Zhou et al.  have integrated ion exchange membranes and electric field for protein cleanup and enrichment. The entire analysis protocol of crude sample, including desalting, enrichment of proteins, and identification or quantification by ESI-MS, could be completed within 20 min. Under electric field, ion exchange membrane could adsorb multiple layers of proteins, exceeding the capacity limit of common membrane. The high trapping ability of ion exchange membrane makes it a good candidate as the support for enzyme immobilization.
In this study, a cation-exchange-membrane-based enzyme micro-reactor was designed and applied for protein digestion, in which trypsin was immobilized on the cation exchange membrane by electrostatic and hydrophobic interaction. The enzyme immobilization process could be achieved simply by pumping trypsin solution through the reactor within several minutes, which is much faster than other immobilization methods. After each cycle of protein digestion, the reactor can be easily regenerated to eliminate the cross-interference of different protein samples. The characterization of the proposed reactor was performed in terms of the amount and the activity of immobilized trypsin, and the repeatability. The influences of residence time and protein concentration on the digestion efficiency were also investigated. The membrane micro-reactor was then coupled with LC-MS for online protein digestion, peptide separation and detection. Three model proteins, including cytochrome c (CYC), myoglobin (MYG), and BSA, were selected to test the performance of the coupling system, and the digestion results were compared with those of in-solution digestion, showing that our proposed micro-reactor is more effective online digestion system and can be used as a better alternative to in-solution digestion. The system was also used for the analysis of yeast cell lysate, and 130 proteins were identified with simple sample pretreatment.
Chemicals and reagents
CYC (equine heart) was obtained from Merck (NJ, USA), and MYG (equine) and BSA from Sigma-Aldrich (MO, USA). Trypsin was purchased from Amresco Inc. (OH, USA). N-α-benzoyl-l-arginine ethyl ester (99%, BAEE) and N-α-benzoyl-l-arginine (99%, BA) were from Alfa Aesar (MA, USA). dl-1,4-dithiothreitol (99%, DTT), iodoacetamide (98%, IAA), and trifluoroacetic acid (TFA) were from Acros Organics (NJ, USA). HPLC grade acetonitrile (ACN) and formic acid (FA) were purchased from Dikma Technologies Inc. (CA, USA). Purified water was provided by Wahaha Group Co., Ltd. (Hangzhou, China). All the other chemicals and reagents were of analytical grade. All the reagents were used without further purification.
Preparation of trypsin and protein solutions
The trypsin was freshly prepared in 50 mM Tris/HCl (pH 8.0) containing 20 mM CaCl2. Model proteins (CYC, MYG, and BSA) were respectively dissolved in 50 mM NH4HCO3 and stored at −20 °C. CYC (0.1 mg mL−1) was digested without denaturation. MYG (3 mg mL−1) was first denaturated with 8 M urea for 3 h at 60 °C and then diluted with 50 mM NH4HCO3 to the protein concentration of 0.3 mg mL−1. BSA (3 mg mL−1) was reduced with 15 mM DTT for 1 h at 55 °C, followed by incubation with 25 mM IAA for 30 min at the room temperature in the dark, and sequentially diluted to 0.3 mg mL−1 using 50 mM NH4HCO3. The denaturated MYG and BSA were ready for digestion.
Yeast proteins were extracted by sonicating 40 mg of wet cell pellet in 1 mL of RIPA buffer (25 mM Tris/HCl at pH 7.6, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% sodium deoxycholate, 0.1% SDS, Halt Protease Inhibitor Cocktails (Thermo Scientific, IL, USA) included) five times for 30 s with 1 min on ice between each pulse, followed by centrifugation at 14,000×g for 15 min to pellet the cell debris. The final protein concentration in the supernatant was determined using BCA assay. The yeast proteins (10 mg mL−1, 8 M urea added) were reduced and alkylated as BSA and diluted to 1 mg mL−1 for further digestion.
For in-solution digestion, the trypsin solution was added to 1 mL of protein solution (0.1 mg mL−1 CYC, 0.3 mg mL−1 MYG, 0.3 mg mL−1 BSA, or 1 mg mL−1 yeast proteins) to obtain a trypsin/protein ratio of 1/50 (w/w). The solution was incubated for 6 h (8 h for yeast proteins) at 37 °C, followed by the addition of 25 μL of 10% FA to stop the digestion. The digest was filtered through 0.22 μm cellulose membrane and analyzed by the LC-MS system.
Fabrication and operation of the membrane-based enzyme micro-reactor
The designed enzyme micro-reactor was operated as follows. (a) Trypsin immobilization was achieved by delivering trypsin solution (1 mg mL−1) through the reactor via a syringe pump at the flow rate of 5 μL min−1 for 2 min. And then, 50 mM NH4HCO3 was passed at the same flow rate for 4 min to remove the unimmobilized trypsin. (b) The prepared protein solution was continuously introduced into the reactor at the flow rate of 2 μL min−1. The effluent from the outlet was collected using a sample vial or a sample loop and analyzed by LC-MS. (c) After protein digestion, the reactor was sequentially regenerated by delivering 10% NaCl (containing 1% NaOH), 4% HCl, and water at the flow rate of 10 μL min−1 for 15, 5, and 15 min, respectively. The entire regeneration process costs only 35 min, and then the reactor was ready for the next cycle of operation from (a) to (c).
Characterization of the enzyme micro-reactor
Amount of the immobilized trypsin
To measure the amount of trypsin adsorbed on the membrane, trypsin solution (1 mg mL−1) was passed through the reactor at the flow rate of 5 μL min−1 for 2 min, and the effluent from the outlet end was completely collected. The trypsin solution (1 mg mL−1) and the effluent were both diluted by 10-fold with 50 mM Tris/HCl (pH 8.0, containing 20 mM CaCl2) and analyzed by LC-MS, respectively. The extracted ion chromatograms of trypsin (m/z 1,942.17; Z, +12) were extracted and integrated, and the trypsin concentration in the effluent was calculated based on the peak areas of trypsin in both solutions. The amount of immobilized trypsin was determined by the decreased trypsin amounts of three repeated measurements.
Assay of the enzyme activity
The activity of free trypsin in solution was also determined for comparison with that of the immobilized trypsin. Twenty microliters of trypsin solution (0.1 mg mL−1) were mixed with 1 mL of BAEE solutions at the above concentrations, respectively, and then the solutions were incubated for 10 min at 37 °C. The reactions were stopped by the addition of 20 μL of 20% FA. After the filtration through 0.22 μm cellulose membrane, the solutions were analyzed by LC-UV.
Online coupling of the enzyme micro-reactor with LC-MS for protein identification
The membrane-based enzyme micro-reactor was online connected to LC-MS via a six-port switching valve with a 20-μL PEEK sample loop, as shown in Fig. 1b. The entire protein analysis process, including protein digestion, peptide desalting, separation and detection, could be achieved in three steps. The first step was the immobilization of trypsin according to the above operation procedure when the valve was at position 1. After switching to position 2, the prepared protein solution was continuously pumped at the flow rate of 2 μL min−1 for 25 min, and the effluent was collected in the sample loop. In the third step, by switching the valve back to position 1, the collected digests were transferred to the separation column. These produced peptides were first concentrated and desalted using the initial gradient with low percentage of organic solvent, and then separated with an optimized elution program. At the same time, the enzyme reactor was regenerated for the analysis of another protein sample.
LC and MS conditions
All the LC-MS experiments were performed on an Agilent 1200 series HPLC system coupled with Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight mass spectrometer (Agilent Technologies, CA, USA). A Zorbax 300SB-C18 column (150 × 2.1 mm (i.d.) and 5 μm particle size; Agilent Technologies, CA, USA) was utilized for peptide separation. The mobile phase consisted of water (containing 0.1% FA, solvent A) and ACN (containing 0.1% FA, solvent B), and the flow rate was 0.2 mL min−1. The optimized gradient for the digest of CYC was: 0–15 min, 5% B; 15–65 min, 5–60% B; 65.5–80 min, 95% B; and 80.5–95 min, 5% B. For MYG and BSA, the elution program was adjusted as follows: 0–15 min, 5% B; 15–90 min, 5–60% B; 90.5–105 min, 95% B; and 105.5–120 min, 5% B. For yeast cell lysate, the gradient was: 0–15 min, 5% B; 15–200 min, 5–50% B; 200–230 min, 50–100% B; 230–240 min, 100% B; and 240.5–255 min, 5% B.
The effluent during first 15 min was delivered to waste so that the salt in the effluent would not contaminate the ion source of MS. The Agilent Jet Stream ESI source was operated in the positive mode and the experimental parameters were set as follows: nebulizer, 40 psig; dry gas temperature, 325 °C; dry gas flow, 10 L min−1; sheath gas temperature, 325 °C; sheath gas flow, 10 L min−1; capillary entrance voltage, 3,500 V; fragmentor, 175 V; and skimmer1, 65 V. The QTOF MS was tuned and calibrated with Tuning Mix (Agilent Part Number G1969-85000). The auto MS/MS mode was used for the analysis of protein digest with fixed collision energy of 23 V. The MS and MS/MS data were acquired at the same rate of 1.03 spectra s−1 in the same mass range of m/z 100–2,000 for CYC and m/z 100–3,000 for MYG, BSA, and yeast cell lysate, and the m/z values were corrected by two ions (m/z 121.0509 and 922.0098 in the reference mass solution, Agilent Part Number G1969-85000) to ensure the mass error within 2 ppm.
The obtained MS and MS/MS data of CYC, MYG, and BSA were first analyzed by MassHunter Qualitative Analysis B.02.00 (Agilent Technologies, CA, USA) to extract compounds from the total ion chromatograms, and then submitted to Mascot (available at www.matrixscience.com) for protein identification. In “Peptide Mass Fingerprint” search, the “Peptide tol.” was set to ±0.02 Da with “oxidation (M)” as the variable modification. And for “MS/MS Ions Search”, the “Peptide tol.” and “MS/MS tol.” were set to ±0.02 and 0.01 Da, respectively. When the BSA digest data were searched, the “carbamidomethyl (C)” was also selected as the fixed modification. The MS/MS data of yeast cell lysate were analyzed by Spectrum Mill (Agilent Technologies, CA, USA) for protein identification.
Results and discussion
Characterization of the enzyme micro-reactor
Typically, the enzyme immobilization with tedious procedure, such as covalent binding or layer-by-layer adsorption, would take tens of minutes or even several hours. In our design, the immobilization process was rapid, simple, and mild, which could be achieved in 2 min by simply pumping trypsin solution through the reactor. Dissolved in Tris/HCl at pH 8.0, trypsin (pI = 10.5) was positively charged and could bind on the cation exchange membrane through electrostatic interaction. In addition, the PS-DVB matrix of the membrane could also adsorb trypsin by hydrophobic interaction. The electrostatic interaction was proved to dominate the immobilization process, evidenced by the fact that the reactor preserved the digestion ability after being flushed with 80% ACN for 1 h.
The micro-channel on the PEEK membrane, serving as the reaction chamber, was etched through the membrane so that both the upper and the under layer cation-exchange-membranes could be used to carry trypsin. The choice of PEEK as the material for both the micro-channel and the connecting capillaries is because the nonspecific adsorption of trypsin and proteins could be minimized. Moreover, the trypsin and the protein solution were delivered parallel to the membrane surface, not penetrating through the membrane as did in the reported membrane reactors [10–14], resulting in very low back pressure even at a relatively high flow rate.
Repeatability and reusability
Effect of digestion time and protein concentration on digestion efficiency
Effect of the digestion time and the protein concentration on the digestion efficiency of CYC by the membrane enzyme micro-reactor (n = 3)
Number of matched peptides
Sequence coverage (%)
Digestion time (s)
14 ± 1
73 ± 1
11 ± 1
68 ± 3
9 ± 1
63 ± 2
Protein concentration (mg mL−1)
14 ± 1
73 ± 1
7 ± 1
47 ± 4
6 ± 1
48 ± 1
4 ± 1
41 ± 7
5 ± 1
48 ± 2
The effect of the protein concentration was also tested using CYC as model protein with the digestion time of 14.4 s, and 3 μL of the product was analyzed. As shown in Table 1, the peptide number and the sequence coverage decreased significantly to the average values of 4% and 41%, respectively, as the CYC concentration reduced to 0.005 mg mL−1(corresponding to ~1.3 pmol CYC). The possible explanation was the nonspecific adsorption of proteins and produced peptides on the cation-exchange membranes, which might become an obvious factor that affected the digestion efficiency for low abundant proteins. One solution was to increase the amount of immobilized enzyme on the membranes so that the nonspecific adsorption could be inhibited. Additionally, the increased enzyme amount could enhance the reaction velocity as well. When the enzyme immobilization time was prolonged to 20 min, higher sequence coverage of 48% with better repeatability was obtained in our experiments. But when 0.001 mg mL−1 CYC was digested, no matched peptide was detected. To improve the sensitivity of the reactor, upstream protein enrichment technologies were necessary. Fortunately, the reactor could be easily integrated with on-line enrichment technologies (e.g., C18 trap column) through a commercial switching valve with a sample loop. After the enrichment of low abundant proteins on the trap column, the trapped proteins could be eluted into the sample loop, and then delivered by the digestion buffer to the reactor for digestion. The elution solvent and the volume of the sample loop might require optimization to obtain a high protein recovery, but this method could provide a simple and automatic solution for the reactor to analyze low abundant proteins.
Online coupling of the enzyme micro-reactor with LC-MS for protein analysis
An integrated platform for protein analysis was established by online combining the micro-reactor with LC-QTOF MS system. The protein digest was totally collected by a 20-μL sample loop and then transferred to the C18 separation column without any loss or contamination, ensuring the accuracy of analysis results. Besides, the peptides were first concentrated and desalted on the C18 column for better separation and no contamination to the ion source. The regeneration and the trypsin immobilization of the reactor, taking about 45 min in total, could be achieved during the peptide separation process.
Digestion of three model proteins through the membrane-based enzyme micro-reactor and in-solution digestion
In-solution digestion (6 h)
Digestion by reactor (14.4 s)
Sequence coverage (%)
Sequence coverage (%)
A novel membrane-based enzyme micro-reactor was presented by immobilizing trypsin on the cation exchange membrane via the electrostatic and hydrophobic interaction. The immobilization process was rapid and mild with the enzyme activity maximumly preserved. The cross-interference of different protein samples could be completely eliminated by regenerating the reactor every time after digestion, ensuring more reliable results of protein identification. The membrane reactor was coupled online with LC-MS for the digestion of CYC, MYG, and BSA as model proteins, showing better sequence coverage than that obtained from 6-h in-solution digestion and much shorter operation time. And 130 proteins were identified when yeast cell lysate was analyzed using this system with simple sample pretreatment. The established membrane-reactor-LC-MS system, involving on-line protein digestion and peptide mapping, provided an automatic and efficient platform for protein analysis.
This work was financially supported by the National Natural Science Foundation of China (grant no. 20975005 and 21027012) and the Fundamental Research Funds for the Central Universities. We thank Dr. Peng Chen and Ph.D. student Jie Li from Peking University for providing the yeast cell lysate.