Up to now, gel electrophoresis remains the most powerful

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1 pubs.acs.org/ac Polyacrylamide Gel with Switchable Trypsin Activity for Analysis of Proteins Fangjie Liu,, Mingliang Ye,*, Chunli Wang,, Zhengyan Hu,, Yi Zhang,, Hongqiang Qin,, Kai Cheng,, and Hanfa Zou*, CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China Graduate School of Chinese Academy of Sciences, Beijing , China *S Supporting Information ABSTRACT: Trypsin was immobilized on a variety of materials to improve digestion efficiency. However, because the immobilized trypsin will digest proteins during electrophoresis, direct immobilization of active trypsin in polyacrylamide gel will compromise the protein separation. To overcome this problem, here we report a novel polyacrylamide gel with switchable trypsin activity. It was prepared by copolymerization of the PEG trypsin aprotinin complex during the gel-casting step. Because the inhibitor aprotinin binds strongly with trypsin at alkaline ph, this novel gel does not display hydrolytic activity during electrophoresis. After electrophoresis, the activity of trypsin embedded in gel could be recovered by simply washing away the bound inhibitor at a low ph. It was demonstrated that this unique switchable activity design allowed high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis. Up to now, gel electrophoresis remains the most powerful protein separation technology for proteomics. The twodimensional gel electrophoresis (2DE) enables effective resolution of more than 5000 proteins. 1 Combined with mass spectrometry, it is an indispensible complement of shot-gun proteomics for protein analysis. 2 To identify proteins in the gel, gel-separated proteins are first excised and digested by a sequence-specific protease such as trypsin into peptides, and then the proteolytically derived peptides were eluted for MS analysis. 3 To digest the proteins in the gel slices, the protease outside must come into contact with the separated proteins in the gel, which is often achieved by incubating the dehydrated gel slices with buffer-containing protease in the classical in-gel digestion protocol. 4 Most of the components in the buffer will diffuse into the gel network, provided that the components are tiny enough. Unfortunately, the protease is not always small enough. Some useful proteases such as the Glu-C protease (MW 29 kda) are not efficient for in-gel digestion because their sizes are above the limit (25 kda) of efficiently entering in the gel. 5 Trypsin (MW 24 kda) is relatively small and therefore generally used for the in-gel digestion procedure. However, the gels for separation of small proteins (<2 kda) are more dense and the trypsin cannot access these proteins either. To overcome the problem of limited accessibility of the protease to the substrate protein, some alternative ways have been developed. 6 On-membrane digestion is one of these methods. By definition, it means that the proteins entangled in the gel matrix can be transferred to blotting membranes and were then directly digested on the membranes. However, considering that the blotting membranes are originally produced to adsorb proteins, the proteases introduced to perform the digestion and the digested peptide will be adsorbed as well. 5 To overcome this limitation, high concentrations of acetonitrile were often used; however, this method cannot lead to high recovery of large and hydrophobic peptides. 7 Another approach to digest proteins adsorbed on the membrane is to dissolve the membranes using some organic solvents prior to digestion Though many efforts have been made to improve this method, its performance is still not good enough because of the partial solubility of proteins or peptides in this system for dissolving membranes. Another interesting approach used to overcome the protease accessibility problem is to use reversible polyacrylamide gels. 11 After electrophoresis, the filament frame of this type of gel could be conditionally cleaved to expose the proteins inside. Whereas this method directly counteracted the problem of low enzyme accessibility, it was not widely utilized as expected probably because of its difficulty in gel preparation and poor resolution. 5 Trypsin has been immobilized on a number of matrices for protein digestion in MS-based protein analysis This approach has many advantages, including highly efficient digestion rate, reduced autolysis products, etc. Can we immobilize trypsin on the polyacrylamide gel for electrophoresis of proteins? To prepare such a gel is not difficult. The key issues include the following: (1) there should be no Received: June 13, 2013 Accepted: July 16, 2013 Published: July 16, American Chemical Society 7024

2 Scheme 1. Schematic Illustration of the Workflow for Analysis of Proteins by the Trypsin Activity Switchable Gel. a a (A) The gel with embedded trypsin-inhibitor complex, (B) separation of proteins by the novel gel via electrophoresis, and (C) in-situ digestion of separated proteins by the trypsin embedded in the gel after washing away inhibitors. hydrolysis activity in the gel during electrophoresis. (2) The hydrolysis activity could be turned on after electrophoresis. To achieve this purpose, we proposed a promising in situ digestion scheme, as shown in Scheme 1. Trypsin was copolymerized in the polyacrylamide gel during the gel-casting stage. To prevent the possible digestion of proteins by the embedded trypsin during electrophoresis, the hydrolysis activity of trypsin was suppressed by a reversible enzyme inhibitor. After electrophoresis, the hydrolysis activity of trypsin was recovered by removing the inhibitor. This switchable activity design would allow for high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis. EXPERIMENTAL SECTION For casting the gel embedded with trypsin, we simply mixed 100 μl of10μg/μl trypsin (in 1 mm HCl aqueous solution) with the other components, 2.4 ml Milli-Q H 2 O, 1.25 ml of 40% acrylamide/bis solution (29: 1, Bio-Rad), 1.3 ml of 1.5 M Tris-HCl buffer (ph 8.8), 50 μl of 10% SDS, 50 μl of 10% ammonium persulfate, and 3 μl of TEMED (Table S1 of the Supporting Information), and then we left it to polymerize at room temperature. As for other SDS PAGE gradient gels, including the conventional gels and the gels embedded with the trypsin aprotinin and PEG trypsin aprotinin complexes, they were prepared in the same way (see the Supporting Information). To monitor the trypsin activity of the prepared gels with trypsin, a gel slice ( cm 2, 2 μg trypsin) was excised and cut into pieces in 1 ml of 50 mm NH 4 HCO 3 buffer in an Eppendorf tube. The synthetic peptide (2 μg) was then added to the above tube for digestion at 37 C for about 1 h. Hydrolysis products were then detected by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). For the analysis of HeLa proteins by the gel embedded with the PEG trypsin aprotinin complex, we loaded 16 μg of HeLa proteins onto each lane. After electrophoresis, the gel was stained by the Coomassie Brilliant Blue R-250 dye. It was destained by aqueous solution containing 50% ethanol and 10% acetic acid, and then the gel was cut into eight slices. The destaining solution also washed the inhibitor aprotinin away. The buffer with gel slices was then switched with the 50 mm NH 4 HCO 3 buffer for in situ digestion overnight at 37 C. The resulting digests were then analyzed by LC MS/MS (see the Supporting Information). 7025

3 Analytical Chemistry Figure 1. Monitor the trypsin activity by MALDI-TOF-MS. (A) Direct analysis of the synthetic peptide (VIFIEHAKRKG, MW Da), incubation of the synthetic peptide with (B) the blank gel without trypsin, (C) the gel with embedded trypsin, (D) the gel with the embedded trypsin aprotinin complex; (E) the same gel with (D) except with the removal of aprotinin with vigorous washing at low ph. Figure 2. Separation of protein standards by the (A) gel with embedded trypsin in the presence of 0.3% SDS, (B) gel with the embedded trypsin ABA complex, (C, D) gel with the embedded trypsin aprotinin complex, (G) gel with the embedded PEG trypsin aprotinin complex, (E, F, H) parallel control gels without trypsin. (A, B, C, E) were stained by Coomassie; (D, F, G, H) were stained by silver. Markers 1 and 2 had a prestained protein ladder of 10 recombinant proteins and a prestained protein molecular weight marker of 6 natural proteins, respectively. RESULTS AND DISCUSSION For separation by SDS PAGE, a high concentration of SDS (0.3% SDS) could be added in the running buffer for separation of proteins. The trypsin may lose its activity under this condition. After separation, the trypsin may gain its activity when the SDS is removed from the gel by repeated washing. If the above assumption were true, then a simple solution could be developed to control the activity of trypsin embedded in the gel without any additional inhibitors. Thus, we first tested if the trypsin still had activity when they were entrapped in the gel with the presence of 0.3% SDS. The gel slice was incubated with the synthetic peptide to detect the trypsin activity. A control experiment was performed as identical to above, except that a gel slice from a SDS PAGE gel without trypsin was used. Figure 1 gives the MALDI-TOF-MS spectra for analysis of the products. Two main hydrolysis product peaks of and were observed for the gel slice with copolymerized trypsin (Figure 1C), while only one peak at (Figure 1B) corresponding to the intact peptide (Figure 1A) was observed for the gel without trypsin. This indicated that trypsin encapsulated in the hydrogel network still has hydrolysis activity. Thus the SDS presented in the gel cannot diminish its activity. We also applied the prepared gel containing trypsin for electrophoresis of protein mixture, however clear protein bands were barely observed after electrophoresis (Figure 2A). This was not surprising since the trypsin entrapped in the gel still 7026

4 Scheme 2. Schematic Illustration for the Coupling of Trypsin to PEG Macromolecule Figure 3. Separation of protein standards by the (A) gel with the embedded PEG trypsin aprotinin complex and the (B) control gel without trypsin. Fractionation of total HeLa cell lysate by the gel with the PEG trypsin aprotinin complex. (C) SDS PAGE image stained by Coomassie, and (D) the number of peptides identified from each fraction. preserved hydrolysis activity, which would inevitably cut the proteins during electrophoresis. Therefore, without the addition of an additional inhibitor, the gel with switchable trypsin activity cannot be prepared. P-aminobenzamidine (ABA) is a popular inhibitor of trypsinlike serine proteases. We tried to use ABA to prepare such a gel. However, when the ABA hydrochloride was added with trypsin to the polymerization solution, precipitation was observed. This was because a neutral complex was formed when ABA, a cation, mixed with the anionic surfactant SDS. We attempted to use the prepared gel for separation of proteins. Clear protein bands were not obtained either (Figure 2B). We then turned to test another trypsin inhibitor, aprotinin. It is a small polypeptide with a MW of 6512 that can reversibly couple to trypsin with a 1: 1 stiochiometry to form a complex. 15 This complex was very stable over a narrow ph range centered around ph 8.3 and would be dissociated only when the ph was lower than 3. This unique ph dependent association/ dissociation characteristic between trypsin and aprotinin makes aprotinin a perfect inhibitor for preparation of the activity switchable gel. To test the inhibition and recovery of the trypsin activity, two gel slices with the same size were excised from the prepared gel containing the complex. One gel slice was directly incubated with the synthetic peptides in the NH 4 HCO 3 buffer. No hydrolysis products were observed, indicating that no trypsin activity was presented in the gel (Figure 1D). Another gel slice was washed by 1% acetic acid solution (ph < 3) to remove aprotinin. It was found that the 7027 substrate peptide was digested, which indicated that trypsin recovered its hydrolysis activity without a hitch (Figure 1E). The washing at low ph led to the dissociation of the complex, and because aprotinin was relatively small (MW 6512 Da), it could easily get out of the gel, leaving trypsin alone in the gel network. Once the ph was changed back to around ph 8.0, the gel slice displayed a predominant hydrolysis activity. In this way, the activity of trypsin entrapped in the gel can be switched off and then on as required. The prepared trypsin aprotinin gel was applied for separation of protein standards (i.e., the prestained protein ladder). Compared with that in Figure 2 (panels A and B), clear protein bands were observed for this gel, as shown in Figure 2 (panels C and D). More importantly, the separation of these proteins were as good as that of the control experiment using the gel without trypsin (Figure 2, panels E and F). The above data indicated that the presence of the complex in the gel did not deteriorate the separation. However, it could be seen that the background for the trypsin aprotinin gels were not homogeneous. For both silver and Coomassie staining gels, the bottom sections were much darker than the top sections (Figure 2, panels C and D). This indicated that the trypsin aprotinin embedded in the gel probably moved down in the electric field during the electrophoresis. To prepare a gel with homogeneous trypsin activity, the movement of the complex in the gel during electrophoresis must be prevented. To prevent the mobility of the complex, we attempted to increase its size by coupling the trypsin to the PEG

5 macromolecule, as shown in Scheme 2. The hydrophilic PEG macromolecule has 4-arms, and on every arm there was a reactive group N-hydroxy succinimide (NHS) ester, which could readily react with the primary amines. The obtained gel was applied to separation of protein standards followed by staining with silver. It was found that the background of the gel was homogeneous in color, indicating that trypsin movement during SDS PAGE was prevented (Figure 2, panels G and H). Compared with the control gel, the gel embedded with the PEG trypsin aprotinin complex unexpectedly preserved favorable separation performance, or even better, because each band of protein mixture was far apart from the others (Figure 3A). Due to its robust separation performance, SDS PAGE was often used for fractionation of proteins in proteomics analysis. 4 We then applied the casted SDS PAGE system for analysis of the complex HeLa cell protein. The high performance of this gel was confirmed by the good separation of protein markers. The digests from each fraction (Figure 3C) were subjected to LC MS analysis performed on a LTQ linear mass spectrometer. On average, about eight hundreds peptides were identified from each fraction (Figure 3D). With consideration that only about 2 μg of proteins were present in a gel slice and only half were loaded for LC MS/MS analysis, the above numbers were excellent. To make sure the peptides identified from each fraction were due to in situ digestion, a control experiment was performed as above, except that a gel without any trypsin was used. It was found that almost no peptide was identified, which indicated that the proteins in the gel were successfully digested in situ by the trypsin immobilized in the gel. Similar numbers of peptides identified from the early fractions and later fractions indicated that the hydrolysis activity was present throughout the gel, which confirmed that the trypsin complex did not move down the gel during electrophoresis. The above results clearly indicated that the prepared gel with switchable trypsin activity was readily applied to the analysis of complex protein samples. CONCLUSIONS A novel gel with immobilized trypsin was prepared by embedment of a PEG trypsin aprotinin complex in the gel network during the gel-casting step. The big size of the complex prevented the movement of trypsin in the electric field during electrophoresis. The complex of trypsin with an inhibitor allowed the separation of proteins by electrophoresis without any risk of digestion. After removing the inhibitor at low ph, in situ digestion of proteins in the gel was successfully achieved without additional trypsin. This switchable activity design allowed high resolution of complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis. As a prototype gel system with switchable protease activity, its performance is expected to be improved by further optimization. ASSOCIATED CONTENT *S Supporting Information Detailed experimental procedures, lists of components, and amounts acquired for gel preparation. This material is available free of charge via the Internet at AUTHOR INFORMATION Corresponding Author *H.F.Z.: , hanfazou@dicp.ac.cn; tel, ; fax, M.L.Y.: , mingliang@dicp.ac.cn; tel, ; fax, Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS Financial support from the Creative Research Group Project of NSFC (Grant ), the National Natural Science Foundation of China (Grants and ), the China State Key Basic Research Program Grant (Grants 2012CB910101, 2013CB911202, and 2012CB910604), National Key Special Program on Infection Diseases (Grant 2012ZX ), and the Analytical Method Innovation Program of MOST (Grant 2012IM030900) are grateful acknowledged. REFERENCES (1) Gorg, A.; Weiss, W.; Dunn, M. J. Proteomics 2004, 4, (2) Aebersold, R.; Mann, M. Nature 2003, 422, (3) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, (4) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. Nat. Protoc. 2006, 1, (5) Rabilloud, T.; Vaezzadeh, A. R.; Potier, N.; Lelong, C.; Leize- Wagner, E.; Chevallet, M. Mass Spectrom. Rev. 2009, 28, (6) Lahm, H. W.; Langen, H. Electrophoresis 2000, 21, (7) Bunai, K.; Nozaki, M.; Hamano, M.; Ogane, S.; Inoue, T.; Nemoto, T.; Nakanishi, H.; Yamane, K. Proteomics 2003, 3, (8) Luque-Garcia, J. L.; Zhou, G.; Sun, T. T.; Neubert, T. A. Anal. Chem. 2006, 78, (9) Luque-Garcia, J. L.; Zhou, G.; Spellman, D. S.; Sun, T. T.; Neubert, T. A. Mol. Cell. Proteomics 2008, 7, (10) Lin, Y.; Li, Y.; Liu, Y.; Han, W. J.; He, Q. Z.; Li, J. L.; Chen, P.; Wang, X. C.; Liang, S. P. Electrophoresis 2009, 30, (11) Bornemann, S.; Rietschel, B.; Baltruschat, S.; Karas, M.; Meyer, B. Electrophoresis 2010, 31, (12) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, (13) Samanta, B.; Yang, X. C.; Ofir, Y.; Park, M. H.; Patra, D.; Agasti, S. S.; Miranda, O. R.; Mo, Z. H.; Rotello, V. M. Angew. Chem., Int. Ed. 2009, 48, (14) Lee, J.; Lee, Y.; Youn, J. K.; Bin Na, H.; Yu, T.; Kim, H.; Lee, S. M.; Koo, Y. M.; Kwak, J. H.; Park, H. G.; Chang, H. N.; Hwang, M.; Park, J. G.; Kim, J.; Hyeon, T. Small 2008, 4, (15) Raspi, G.; Lo Moro, A.; Spinetti, M.; Molinari, M. The Analyst 1989, 114,