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DOI 10.1002/pmic.201300487
TECHNICAL BRIEF
Hydrophilic polydopamine-coated magnetic graphene nanocomposites for highly efficient tryptic immobilization Chenyi Shi1 , Chunhui Deng1 , Yan Li2∗ , Xiangmin Zhang1 and Pengyuan Yang1 1 2
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, P. R. China Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai, P. R. China
In this work, polydopamine-coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as-made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsinimmobilized MG@PDA (MG@PDA-trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA-trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA-trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC-ESI-MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16-h in-solution digestion (264 proteins), indicating the great potential of MG@PDA-trypsin nanocomposites as the supports for high-throughput proteome study.
Received: November 4, 2013 Revised: January 10, 2014 Accepted: April 1, 2014
Keywords: Digestion / Immobilization of trypsin / Magnetic polydopamine-coated magnetic graphene nanocomposites / MALDI-TOF MS / Nanoproteomics
Additional supporting information may be found in the online version of this article at the publisher’s web-site
With the development of post-genomic time, proteomics has drawn an on-going worldwide research attention over the past decades [1, 2]. One of its main challenges is to develop efficient and rapid techniques for identification of various proteins encoded by the genomes. Up to now, MS has emerged Correspondence: Dr. Chunhui Deng, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, P. R. China E-mail: [email protected] Fax: +86-21-65641740 Abbreviations: DOPA, dopamine; IR, infrared spectroscopy; MG@PDA, polydopamine-coated magnetic graphene; SEM, scanning electron microscopy; TEM, transmission electron microscopy C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
as the most straightforward and powerful tool for protein identification and characterization [3, 4]. Proteins are usually digested and separated into peptides by proteases. And then obtained digests were performed by MS and processed subsequent database researching [5, 6]. In this peptide-based strategy, it is vital of rapid and complete digestion of all proteins to the high throughput and accuracy identification of proteins. In proteomics analysis, the traditional in-solution digestion of proteins is typically performed by trypsin for 12–16 h at 37⬚C with a weight ratio of trypsin-to-substrate ∗ Additional corresponding author: Dr. Yan Li, E-mail: [email protected] Colour Online: See the article online to view Scheme 1 and Figs. 2–4 in colour.
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typically 1:20–1:40 [7], which makes the whole digestion process time-consuming and labor-intensive. Moreover, the autolysis of enzyme during digestion process may not only suppress the MS signals of proteins, but also generate fragments to interfere with protein sequencing [8,9]. Thus it is an urgent priority to explore more rapid and complete means of enzymatic digestion of proteins, which allows a highly efficient proteolysis for the MS peptide mapping. In recent years, tremendous efforts have been made to immobilize enzyme owing to its distinct merits, such as higher enzyme concentration in limited space leading to shorter digestion time, better stability, reusable, separable from the reaction media and leaving little autolysis byproducts [10]. In addition, immobilized enzymes can be readily connected to separation and identification systems, which are capable of rapid, high-throughput, efficient, and automated proteome analysis [11]. Until now, a variety of materials have been investigated as supports for the immobilization of enzymes, such as membrane, packed beads, capillary, porous polymer monoliths, porous silicon, nanomaterials, sol-gel supports, and hybrids [12–19]. Compared with other supports, magnetic microspheres have unique superiority, such as reusability, ease of manipulation, and lower capacity of nonspecific protein biding [20]. In addition, the magnetic responsive property of magnetic microspheres makes them easy to be separated from the digested peptide solution with a magnetic field, resulting in a simple, fast, and highly effective treatment procedure. Therefore, magnetic microspheres can be applied as an ideal support for enzyme immobilization and have been widely applied in proteomics research in recent years [21]. However, despite the rapid development of magnetic microspheres-based digestion methods, two main problems still remain and obstruct their further application in high-throughput protein profiling. One problem is that the low surface areas of pristine magnetic microspheres may limit the immobilization amount of enzymes [22]. The other is that the poor hydophilicity of the magnetic microspheres will not only hinder the immobilization of hydrophilic trypsin (the most commonly used protease in proteomics study) but will also result in high peptide residue. To solve the first problem, magnetic particles with high-interaction sites or nanocomposites of magnetic microspheres with other components which can offer larger surface area were prepared to improve the loading capacity for enzyme. [23, 24] Graphene, provided with unusual physical and chemical properties, has sparked considerable interest in the recent years [25]. The ultrahigh surface area (2630 m2 /g compared to 10 m2 /g of graphite and 1315 m2 /g of nanotubes) [26] enables graphene to support a high loading capability, which makes it a promoting substrate to combine with magnetic microspheres for a high amount of enzyme immobilization [27]. To improve the hydrophilicity of the magnetic nanocomposites, one promising strategy is to employ a polydopamine (PDA) shell which can be coated on a variety of substrates via the oxidative self-polymerization of dopamine (DOPA) in a C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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basic solution [28]. It has been proven that PDA has several advantages, such as good biocompatibility, excellent environmental stability, and splendid hydrophilicity, which are in favor of enzyme immobilization and protein digestion. The catechol and quinone functional groups, present in the PDA coating, are capable of covalent coupling to amine groups [29], and thus make it easy to directly covalently bond trypsin without the need for introducing any other coupling reagents. The aim of this study was to explore the feasibility of preparing nanocomposites which will combine the advantages of magnetic microspheres, graphene and PDA, and highly efficient tryptic immobilization by means of them. So a highly efficient in situ digestion platform was developed based on these PDA-coated magnetic graphene (MG@PDA) nanocomposites. Due to the large surface area, good hydrophilicity, and strong magnetic response, the prepared nanocomposites possessed the advantages of high trypsin immobilization capacity, low peptide residue, and easy operation, which were successfully applied to achieve high efficiency and highthroughput proteome digestion. The synthetic protocol of the MG@PDA nanocomposites is presented in Scheme 1A. First, graphene was acidified by HNO3 to create carboxylic groups on the outer surface of graphene and make it negatively charged. The acidified graphene could then combine with magnetic microsphere easily via a simple hydrothermal reaction. Thereafter, the obtained magnetic graphene (MG) was modified through the polymerization of DOPA in an alkaline solution (10 mM Tris, pH 8.5) [30]. The as-synthesized product of each step was characterized by different techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and infrared spectroscopy (IR). The morphologies of MG and MG@PDA nanocomposites were characterized by SEM and TEM, respectively. As shown in Fig. 1, the pristine graphene exhibits clean surfaces of a flake-like shape with transparent silk waves (Fig. 1A). After combining with magnetic microsphere, it is observed that magnetic beads with sizes of around 200 nm are loaded on the surface of graphene sheet, which reveals the successful synthesis of MG (Fig. 1B). After coating with PDA, the obtained MG@PDA nanocomposites show a clearly visible layer of PDA with thickness of 10 nm and no free PDA particles are observed in the TEM image, which suggests the successful polymerization of DOPA on the surface of MG. The Raman spectra of graphene and MG@PDA nanocomposites are shown in Supporing Information Fig. 1. As shown in curve a, the characteristic peaks attributed to the structure of graphene are 1368/cm (D-mode), 1596/cm (G-mode), and 2712/cm (2D-mode). After combining with magnetic microspheres, the characteristic peaks at 424/cm and 712/cm assigned to magnetic microspheres indicate the Fe-O vibration (curve b). After coating with PDA, the characteristic peaks at 1297/cm and 1543/cm are assigned to the structure of PDA, which are stretching and deformation of catecholos (curve b) [31, 32]. In the Raman spectrum of MG@PDA nanocomposites (curve b), the peaks of D-mode and G-mode attributed www.proteomics-journal.com
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Scheme 1. (A) The synthetic procedure of MG@PDA nanocomposites. (B) The procedure of proteins digestion by MG@ PDA-trypsin nanocomposties.
to graphene are slightly broadened and the peak of 2D-mode attributed to graphene is greatly decreased, which implies that the Raman spectrum of MG@PDA nanocomposites shows the sum features of graphene and PDA for the characteristic peaks of graphene and PDA are nearly at the same positions.
In order to investigate the hydrophilicity of MG@PDA nanocomposites, graphene and MG@PDA nanocomposites were dispersed in water, respectively. Both dispersions were ultrasonicated for 5 min and then stored for a week without agitation. As shown in Fig. 2, graphene precipitates out of the water quickly in a couple of minutes. However, owing to
Figure 1. SEM images of (A) graphene and (B) MG nanocomposites; TEM images of (C) and (D) MG@PDA nanocomposites.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. The dispersions of graphene and MG@ PDA nanocomposites at different periods of time.
abundant hydroxyl and amine groups on PDA shells, the dispersion of MG@PDA nanocomposites remains stable even after one-week storage indicating the excellent hydrophilicity of the nanocoposites which makes it an ideal candidate as a support to immobilize hydrophilic trypsin. It has been proven that PDA is an effective platform for trypsin immobilization. Quinone groups in PDA layer generated from self-oxidation of DOPA can actively react with amino functional groups of trypsin via Michael Addition and/or Schiff base reactions, which leads to trypsin being immobilized on the surface of PDA [33]. In this work, conjugation of trypsin to MG@PDA nanocomposites was accomplished by simply dispersing the nanocomposites into a trypsin solution and stirring for 16 h at pH 6.5. Fourier transform infrared spectroscopy was further used to characterize the MG@PDA nanocomposites after trypsin immobilization. As shown in Supporting Information Fig. 2, the peak at 548/cm is assigned to Fe-O stretching of magnetic microspheres. The Fourier transform infrared spectrum also exhibits trypsin absorption features, such as the amide band I and II of trypsin (1574/cm and 1512/cm), N-H (3412/cm) and C-N stretch mode of trypsin (1288/cm), which confirm the successful immobilization of trypsin. The immobilization ability of MG@PDA nanocomposites for trypsin was determined by comparing the UV adsorption value of the supernatant trypsin solution before and after immobilization procedure at the wavelength of 595 nm, and the amount of immobilized trypsin was calculated to be about 0.175 mg/mg. This amount of immobilized trypsin is much higher than those obtained with other reported magnetic particles as matrices [24, 34]. The high trypsin loading amount can be attributed to the unique structure of graphene, twodimensional layers of the sp2 -bonded carbon and the ultrahigh surface area. The digestion procedure is shown in Scheme 1B. In order to evaluate the digestion efficiency of MG@PDA C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. The mass spectra of Cyt-c after (A) in-solution digestion for 16 h, (B) digestion with MG@PDA-trypsin nanocomposites for 10 min; the mass spectra of Myo after (C) in-solution digestion for 16 h, (D) digestion with MG@PDA-trypsin nanocomposites for 10 min. All matched peptides of Cyt-c and Myo are marked with “C” and “M”, respectively.
trypsin nanocomposites, Cyt-c, and Myo (200 ng/L for each, 50 L) were chosen as standard protein samples. In this study, we choose 10 min as digestion time in order to get the best digestion efficiency of MG@PDA-trypsin nanocomposites within the shortest time without microwave assistance. After 10-min digestion at 37⬚C, the trypsin immobilized nanocomposites were easily retrieved with a magnetic field from solution. For comparison, proteins were also treated by traditional 16-h in-solution digestion at 37⬚C. The digested peptides were analyzed by MALDI-TOF MS, and the results are shown in Fig. 3 and Supporting Information Table 1. According to the results, it is obvious that the digestion efficiency obtained by MG@PDA-trypsin nanocomposites was comparable to that obtained by traditional in-solution digestion, which was owing to the higher enzyme concentration. The sequence coverage obtained from the database of MG@PDA-trypsin nanocomposites is 62% for Cyt-c and 83% for Myo, which is similar to those of other reported www.proteomics-journal.com
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IMERs. For example, in Jiang’s work, the sequence coverage obtained from the database of trypsin-immobilized GO-CONH-Fe3 O4 nanocomposites is 60% for Cyt-c and 84% for Myo [27]. The results indicate that trypsin immobilized MG@PDA nanocomposites are beneficial to improve the digestion efficiency. The digestion efficiency of MG@PDA-trypsin nanocomposites for very small amount of protein with low concentration was investigated. For this purpose, 50 L of 1 ng/L Cyt-c was chosen as a sample. As shown in Supporing Information Fig. 3, after 10-min digestion, six peptides were matched and the corresponding sequence coverage was 39%, which demonstrated the ability of as-made nanocomposites for digestion of low abundance proteins. The non-specific adsorption of peptides on the as-made MG@PDA-trypsin nanocomposites was investigated. First, the MG@PDA-trypsin nanocomposites were employed to digest Myo. After washing with 25 mM NH4 HCO3 for three times, the nanocomposites were used for further digestion of Cyt-c. The results indicate that after digestion of Cyt-c, no peptides of Myo were obtained in the Cyt-c digests while ten tryptic peptides of Cyt-c were matched with the sequence coverage of 60%. The low peptide residue on MG@PDA-trypsin nanocomposites is in virtue of good hydrophile of PDA. The reusability and stability of MG@PDA-trypsin nanocomposites were also evaluated using standard protein Myo as the sample. After five consecutive digestions, the obtained peptides in the fifth time were almost the same as those in the first time (12 common matched peptides), which indicated that the as-made nanocomposites could be reused at least five times with the same effects of digestion. To test the stability of the trypsin immobilized materials, the as-prepared nanocomposites were stored at 4⬚C for one month. The obtained results of digestion were still the same as those obtained by freshly made nanocomposites (sequence coverage of Myo remained as 83%). The above results show the good reusability and stability of MG@PDA-trypsin nanocomposites. The reproducibility of different batches of MG@PDA nanocomposites as well as trypsin immobilization on different batches of MG@PDA nanocomposites were investigated in this study. We repeated the experiments about the reproducibility several times. And all the results are similar to each other. The sequence coverage obtained from the database of MG@PDA-trypsin nanocomposites is around 60% for Cyt-c and 80% for Myo. And the matched peptides were about 11 for Cyt-c and 14 for Myo. The results indicate that the reproducibility of MG@PDA nanocomposites is very good, which enable the pre-synthesized nanocomposites to be an ideal matrix of trypsin immobilization for digestion in complex samples. Encouraged by the high digestion efficiency and good reproducibility of MG@PDA-trypsin nanocomposites, the as-made nanocomposites were further applied in largescale proteomics research. Extracted proteins from skimmed milk were selected to evaluate the digestion capacity for C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. Venn of the overlap of identified proteins from skimmed milk between in solution and MG@PDA-trypsin digestion.
the complex real sample. After digestion at 37⬚C, the obtained peptides were analyzed by nano LC-ESIMS/MS. The Venn of overlap of identified proteins from skimmed milk between traditional in-solution digestion and MG@PDA-trypsin nanocomposites is shown in Fig. 4. As depicted in Supporing Information Table 2 and 3, 321 proteins can be identified with the as-made nanocomposites for 10-min digestion, while only 264 proteins are identified with 16-h in-solution digestion. The overlap is 123 proteins, which is not so large. We speculate the reason why the overlap of identified proteins by MG@PDA-trypsin nanocomposites and in-solution digestion is not so large is relative to the structure and the way for trypsin immobilization of MG@PDAtrypsin nanocomposites, which makes the enzyme digestion sites different from those of in-solution digestion. We chose graphene, the two-dimensional layers of sp2 -bonded carbon, as the substrate to support a high loading capability. Dopamine was employed to provide catechol and quinone functional groups present in the PDA coating, which are capable of covalent coupling to amine groups of trypsin. Thus the structure of graphene and the way for trypsin immobilization with dopamine may result in different enzyme digestion sites between two methods, which are responsible for the non-overlapping nature of in-solution and MA@PDA-trypsin nanocomposites. Although the true reason is not clear, the above results still demonstrate that the MG@PDA-trypsin nanocomposites have high digestion capacity even for complex real samples. The authentic reason is worth to studying in our next work. In summary, magnetic graphene nanocomposites coated with PDA shell were synthesized by a simple approach, trypsin was then immobilized on the surface through PDA chemistry. The as-made nanocomposites were successfully applied in the digestion of proteins. Owing to the large surface of grapheme and high hydrophilicity of PDA, the novel nanocomposites not only have high trypsin immobilization capacity (up to 0.175 mg/mg) which led to a high digestion efficiency of proteins, but also offer a simple sample preparation procedure due to the good magnetic responsiveness of magnetic microspheres. Within 10 min, the peptide sequence coverage of protein was comparable to that obtained by traditional 16-h in-solution digestion. In addition, the asmade nanocomposites exhibited good reusability, stability, www.proteomics-journal.com
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and non-specific adsorption, and were performed well in very small amounts of proteins and complex real samples. Therefore, this strategy offered a novel approach to immobilize trypsin for high digestion efficiency and indicated a great potential for high-throughput proteome profiling. This work was supported by the National Basic Research Priorities Program (2012CB910601 and 2013CB911201), the National Natural Science Foundation of China (21075022, 20875017, and 21105016), Research Fund for the Doctoral Program of Higher Education of China (20110071110007, 20100071120053), Shanghai Municipal Natural Science Foundation (11ZR1403200), and Shanghai Leading Academic Discipline Project (B109). The authors have declared no conflict of interest.
References [1] Kahn, P., From genome to proteome: looking at a cell’s proteinstitle. Science 1995, 270, 369–370. [2] Domon, B., Aebersold, R., Mass spectrometry and protein analysis. Science 2006, 312, 212–217. [3] Jang, H. B., Sung, H. W., Nho, S. W., Park, S. B. et al., Enhanced reliability of avian influenza virus (AIV) and Newcastle disease virus (NDV) identification using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Anal. Chem. 2011, 83, 1717–1725. [4] Spro, J., Sinz, A., A capillary monolithic trypsin reactor for efficient protein digestion in online and offline coupling to ESI and MALDI mass spectrometry. Anal. Chem. 2010, 82, 1434–1443. [5] Ma, J., Zhang, L., Liang, Z., Shan, Y., Zhang, Y., Immobilized enzyme reactors in proteomics. Trends Anal. Chem. 2011, 30, 691–702. [6] Wang, S., Bao, H., Zhang, L., Yuan, P., Chen, G., Infraredassisted on plate proteolysis for MALDI-TOF-MS peptide mapping. Anal. Chem. 2008, 80, 5640–5647. [7] Winter, D., Steen, H., Optimization of cell lysis and protein digestion protocols for the nalysis of Hela S3 cells by LCMS/MS. Proteomics 2011, 11, 4726–4730. [8] Wang, S., Bao, H. M., Yang, P. Y., Chen, G., Immobilization of trypsin in polyanliline-coated nano-Fe3 O4 .carbon nanotube composite for protein digestion. Anal. Chim. Acta. 2008, 612, 182–189. [9] Qiao, L., Liu, Y., Hudson, S. P., Yang, P. Y. et al., A nanoporous reactor for efficient proteolysis. Chem. Eur. J. 2008, 14, 151– 157. [10] Bornscheuer, U. T., Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. 2003, 42, 3336–3337. [11] Ma, J., Liang, Z., Qiao, X., Deng, Q. et al., Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal. Chem. 2008, 80, 2949– 2956.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2014, 14, 1457–1463 [12] Brinkworth, C. S., identification of ricin in crude and purified extracts from castor beans using on-target tryptic digestion and MALDI mass spectrometry. Anal. Chem. 2010, 82, 5246– 5252. [13] Xu, F., Wang, W., Tan, Y., Bruening, M. L., Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion. Anal. Chem. 2010, 82, 10045–10051. [14] Nagai, N., Suzuki, Y., Sekikawa, C., Nara, T. Y. et al., Fabrication of boehmite films with cage-like pores and their properties as enzyme immobilization supports. Mater. Chem. 2012, 22, 3234–3241. [15] Pierre, S. J., Thies, J. C., Dureault, A., Cameron, N. R. et al., Covalent enzyme immobilization onto photopolymerized highly porous monoliths. Adv. Mater. 2006, 18, 1822– 1826. [16] Lin, S., Yao, G., Qi, D., Li, Y. et al., Fast and efficient proteolysis by microwave-assisted protein digestion using trypsinimmobilized magnetic silica microspheres. Anal. Chem. 2008, 80, 3655–5665. [17] Liu, Z., Wang, H. S., Li, B., Liu, C. et al., Biocompatible magnetic cellulose-chitosan hybrid gel microspheres reconstituted from ionic liquids for enzyme immobilization. J. Mater. Chem. 2012, 22, 15085–15091 [18] Dulay, M. T., Baca, Q. J., Zare, R. N., Enhanced proteolytic activity of covalently bound enzymes in photopolymerized sol gel. Anal. Chem. 2005, 77, 4604–4610. [19] Pereira-Medrano, A. G., Forester, S., Fowler, G. J. S., McArthur, S. L., Wright, P. C., Rapid fabrication of glass/PDMS hybrid IMER for high throughput membrane proteomics. Lab Chip 2010, 10, 3397–3406. [20] Gao, J. H., Gu, H. W., Xu, B., Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Accounts Chem. Res. 2009, 42, 1097–1107. [21] Li, Y., Zhang, X. M., Deng, C. H., Functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis. Chem. Soc. Rev. 2013, 42, 8517–8539. [22] Qin, W. J., Song, Z. F., Fan, C., Zhang, W. J. et al., Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18 O labeling and absolute protein quantification. Anal. Chem. 2012, 84, 3138–3144. [23] Huang, J., Li, X., Zheng, Y., Zhang, Y. et al., Immobilization of penicillin G acylase on poly[(glycidyl methacrylate)co-(glycerol monomethacrylate)]-grafted magnetic microspheres. Macromol. Biosci. 2008, 8, 508–515. [24] Deng, Y. H., Deng, C. H., Qi, D. W., Liu, C. et al., Synthesis of core/shell colloidal magnetic zeolite microspheres for the immobilizaton of trypsin. Adv. Mater. 2009, 21, 1377–1382. [25] Novoselov, K. S., Geim, A. K., Morozov, S. V., Jiang, D. et al., Electric field effect in atomically thin carbon films. Science, 2004, 306, 666–669. [26] Stoller, M. D., Park, S. J., Zhu, Y. W., An, J. H., Ruoff, R. S., Graphene-based ultracapacitors. Nano Lett. 2008, 8, 3498– 3502. [27] Jiang, B., Yang, K. G., Zhao, Q., Wu, Q. et al., Hydrophilic immobilized trypsin reactor with magnetic graphene oxide
www.proteomics-journal.com
Proteomics 2014, 14, 1457–1463
1463
as support for high efficient proteome digestion. J. Chromatogr. A 2012, 1254, 8–13.
wall carbon nanohorns and their release. Carbon 2008, 46, 1792–1794.
[28] Lee, H., Dellatore, S. M., Miller, W. M., Messersmith, P. B., Mussel-inspired surface chemistry for multifunctional coatings. Science, 2007, 318, 426–430.
[32] Hong, S., Lee, J. S., Ryu, J., Lee, S. H. et al., Bio-inspired strategy for on-surface synthesis of silver nanoparticles for metal/organic hybrid nanomaterials and LDI-MS substrates. Nanotechnology 2011, 22, 494020.
[29] Lee, H., Scherer, N. F., Messersmith, P. B., Single molecule mechanics of mussel adhesion. Proc. Natl. Acad. Sci. USA 2006, 103, 12999–13003. [30] Yan, Y. H., Zheng, Z. F., Deng, C. H., Zhang, X. M, Yang, P. Y., Facile synthesis of Ti4+ -immobilized Fe3 O4 @polydopamine core-shell microspheres for highly selective enrichment of phosphopeptides. Chem. Commun. 2013, 49, 5055–5077. [31] Fei, B., Qian, B., Yang, Z., Wang, R. et al., Effect of hole size on the incorporation of C60 molecules inside single-
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[33] Lee, H., Rho, J., Messersmith, P. B., Facile conjugation of biomolecules onto surfaces via mussel adhesive protein inspired coatings. Adv. Mater. 2009, 21, 431–434. [34] Li, Y., Yan, B., Deng, C., Tang, J. et al., On-plate digeston of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis. Proteomics 2007, 7, 3661–3671.
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DOI 10.1002/pmic.201300487
TECHNICAL BRIEF
Hydrophilic polydopamine-coated magnetic graphene nanocomposites for highly efficient tryptic immobilization Chenyi Shi1 , Chunhui Deng1 , Yan Li2∗ , Xiangmin Zhang1 and Pengyuan Yang1 1 2
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, P. R. China Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai, P. R. China
In this work, polydopamine-coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as-made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsinimmobilized MG@PDA (MG@PDA-trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA-trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA-trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC-ESI-MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16-h in-solution digestion (264 proteins), indicating the great potential of MG@PDA-trypsin nanocomposites as the supports for high-throughput proteome study.
Received: November 4, 2013 Revised: January 10, 2014 Accepted: April 1, 2014
Keywords: Digestion / Immobilization of trypsin / Magnetic polydopamine-coated magnetic graphene nanocomposites / MALDI-TOF MS / Nanoproteomics
Additional supporting information may be found in the online version of this article at the publisher’s web-site
With the development of post-genomic time, proteomics has drawn an on-going worldwide research attention over the past decades [1, 2]. One of its main challenges is to develop efficient and rapid techniques for identification of various proteins encoded by the genomes. Up to now, MS has emerged Correspondence: Dr. Chunhui Deng, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, P. R. China E-mail: [email protected] Fax: +86-21-65641740 Abbreviations: DOPA, dopamine; IR, infrared spectroscopy; MG@PDA, polydopamine-coated magnetic graphene; SEM, scanning electron microscopy; TEM, transmission electron microscopy C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
as the most straightforward and powerful tool for protein identification and characterization [3, 4]. Proteins are usually digested and separated into peptides by proteases. And then obtained digests were performed by MS and processed subsequent database researching [5, 6]. In this peptide-based strategy, it is vital of rapid and complete digestion of all proteins to the high throughput and accuracy identification of proteins. In proteomics analysis, the traditional in-solution digestion of proteins is typically performed by trypsin for 12–16 h at 37⬚C with a weight ratio of trypsin-to-substrate ∗ Additional corresponding author: Dr. Yan Li, E-mail: [email protected] Colour Online: See the article online to view Scheme 1 and Figs. 2–4 in colour.
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typically 1:20–1:40 [7], which makes the whole digestion process time-consuming and labor-intensive. Moreover, the autolysis of enzyme during digestion process may not only suppress the MS signals of proteins, but also generate fragments to interfere with protein sequencing [8,9]. Thus it is an urgent priority to explore more rapid and complete means of enzymatic digestion of proteins, which allows a highly efficient proteolysis for the MS peptide mapping. In recent years, tremendous efforts have been made to immobilize enzyme owing to its distinct merits, such as higher enzyme concentration in limited space leading to shorter digestion time, better stability, reusable, separable from the reaction media and leaving little autolysis byproducts [10]. In addition, immobilized enzymes can be readily connected to separation and identification systems, which are capable of rapid, high-throughput, efficient, and automated proteome analysis [11]. Until now, a variety of materials have been investigated as supports for the immobilization of enzymes, such as membrane, packed beads, capillary, porous polymer monoliths, porous silicon, nanomaterials, sol-gel supports, and hybrids [12–19]. Compared with other supports, magnetic microspheres have unique superiority, such as reusability, ease of manipulation, and lower capacity of nonspecific protein biding [20]. In addition, the magnetic responsive property of magnetic microspheres makes them easy to be separated from the digested peptide solution with a magnetic field, resulting in a simple, fast, and highly effective treatment procedure. Therefore, magnetic microspheres can be applied as an ideal support for enzyme immobilization and have been widely applied in proteomics research in recent years [21]. However, despite the rapid development of magnetic microspheres-based digestion methods, two main problems still remain and obstruct their further application in high-throughput protein profiling. One problem is that the low surface areas of pristine magnetic microspheres may limit the immobilization amount of enzymes [22]. The other is that the poor hydophilicity of the magnetic microspheres will not only hinder the immobilization of hydrophilic trypsin (the most commonly used protease in proteomics study) but will also result in high peptide residue. To solve the first problem, magnetic particles with high-interaction sites or nanocomposites of magnetic microspheres with other components which can offer larger surface area were prepared to improve the loading capacity for enzyme. [23, 24] Graphene, provided with unusual physical and chemical properties, has sparked considerable interest in the recent years [25]. The ultrahigh surface area (2630 m2 /g compared to 10 m2 /g of graphite and 1315 m2 /g of nanotubes) [26] enables graphene to support a high loading capability, which makes it a promoting substrate to combine with magnetic microspheres for a high amount of enzyme immobilization [27]. To improve the hydrophilicity of the magnetic nanocomposites, one promising strategy is to employ a polydopamine (PDA) shell which can be coated on a variety of substrates via the oxidative self-polymerization of dopamine (DOPA) in a C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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basic solution [28]. It has been proven that PDA has several advantages, such as good biocompatibility, excellent environmental stability, and splendid hydrophilicity, which are in favor of enzyme immobilization and protein digestion. The catechol and quinone functional groups, present in the PDA coating, are capable of covalent coupling to amine groups [29], and thus make it easy to directly covalently bond trypsin without the need for introducing any other coupling reagents. The aim of this study was to explore the feasibility of preparing nanocomposites which will combine the advantages of magnetic microspheres, graphene and PDA, and highly efficient tryptic immobilization by means of them. So a highly efficient in situ digestion platform was developed based on these PDA-coated magnetic graphene (MG@PDA) nanocomposites. Due to the large surface area, good hydrophilicity, and strong magnetic response, the prepared nanocomposites possessed the advantages of high trypsin immobilization capacity, low peptide residue, and easy operation, which were successfully applied to achieve high efficiency and highthroughput proteome digestion. The synthetic protocol of the MG@PDA nanocomposites is presented in Scheme 1A. First, graphene was acidified by HNO3 to create carboxylic groups on the outer surface of graphene and make it negatively charged. The acidified graphene could then combine with magnetic microsphere easily via a simple hydrothermal reaction. Thereafter, the obtained magnetic graphene (MG) was modified through the polymerization of DOPA in an alkaline solution (10 mM Tris, pH 8.5) [30]. The as-synthesized product of each step was characterized by different techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and infrared spectroscopy (IR). The morphologies of MG and MG@PDA nanocomposites were characterized by SEM and TEM, respectively. As shown in Fig. 1, the pristine graphene exhibits clean surfaces of a flake-like shape with transparent silk waves (Fig. 1A). After combining with magnetic microsphere, it is observed that magnetic beads with sizes of around 200 nm are loaded on the surface of graphene sheet, which reveals the successful synthesis of MG (Fig. 1B). After coating with PDA, the obtained MG@PDA nanocomposites show a clearly visible layer of PDA with thickness of 10 nm and no free PDA particles are observed in the TEM image, which suggests the successful polymerization of DOPA on the surface of MG. The Raman spectra of graphene and MG@PDA nanocomposites are shown in Supporing Information Fig. 1. As shown in curve a, the characteristic peaks attributed to the structure of graphene are 1368/cm (D-mode), 1596/cm (G-mode), and 2712/cm (2D-mode). After combining with magnetic microspheres, the characteristic peaks at 424/cm and 712/cm assigned to magnetic microspheres indicate the Fe-O vibration (curve b). After coating with PDA, the characteristic peaks at 1297/cm and 1543/cm are assigned to the structure of PDA, which are stretching and deformation of catecholos (curve b) [31, 32]. In the Raman spectrum of MG@PDA nanocomposites (curve b), the peaks of D-mode and G-mode attributed www.proteomics-journal.com
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Scheme 1. (A) The synthetic procedure of MG@PDA nanocomposites. (B) The procedure of proteins digestion by MG@ PDA-trypsin nanocomposties.
to graphene are slightly broadened and the peak of 2D-mode attributed to graphene is greatly decreased, which implies that the Raman spectrum of MG@PDA nanocomposites shows the sum features of graphene and PDA for the characteristic peaks of graphene and PDA are nearly at the same positions.
In order to investigate the hydrophilicity of MG@PDA nanocomposites, graphene and MG@PDA nanocomposites were dispersed in water, respectively. Both dispersions were ultrasonicated for 5 min and then stored for a week without agitation. As shown in Fig. 2, graphene precipitates out of the water quickly in a couple of minutes. However, owing to
Figure 1. SEM images of (A) graphene and (B) MG nanocomposites; TEM images of (C) and (D) MG@PDA nanocomposites.
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Figure 2. The dispersions of graphene and MG@ PDA nanocomposites at different periods of time.
abundant hydroxyl and amine groups on PDA shells, the dispersion of MG@PDA nanocomposites remains stable even after one-week storage indicating the excellent hydrophilicity of the nanocoposites which makes it an ideal candidate as a support to immobilize hydrophilic trypsin. It has been proven that PDA is an effective platform for trypsin immobilization. Quinone groups in PDA layer generated from self-oxidation of DOPA can actively react with amino functional groups of trypsin via Michael Addition and/or Schiff base reactions, which leads to trypsin being immobilized on the surface of PDA [33]. In this work, conjugation of trypsin to MG@PDA nanocomposites was accomplished by simply dispersing the nanocomposites into a trypsin solution and stirring for 16 h at pH 6.5. Fourier transform infrared spectroscopy was further used to characterize the MG@PDA nanocomposites after trypsin immobilization. As shown in Supporting Information Fig. 2, the peak at 548/cm is assigned to Fe-O stretching of magnetic microspheres. The Fourier transform infrared spectrum also exhibits trypsin absorption features, such as the amide band I and II of trypsin (1574/cm and 1512/cm), N-H (3412/cm) and C-N stretch mode of trypsin (1288/cm), which confirm the successful immobilization of trypsin. The immobilization ability of MG@PDA nanocomposites for trypsin was determined by comparing the UV adsorption value of the supernatant trypsin solution before and after immobilization procedure at the wavelength of 595 nm, and the amount of immobilized trypsin was calculated to be about 0.175 mg/mg. This amount of immobilized trypsin is much higher than those obtained with other reported magnetic particles as matrices [24, 34]. The high trypsin loading amount can be attributed to the unique structure of graphene, twodimensional layers of the sp2 -bonded carbon and the ultrahigh surface area. The digestion procedure is shown in Scheme 1B. In order to evaluate the digestion efficiency of MG@PDA C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. The mass spectra of Cyt-c after (A) in-solution digestion for 16 h, (B) digestion with MG@PDA-trypsin nanocomposites for 10 min; the mass spectra of Myo after (C) in-solution digestion for 16 h, (D) digestion with MG@PDA-trypsin nanocomposites for 10 min. All matched peptides of Cyt-c and Myo are marked with “C” and “M”, respectively.
trypsin nanocomposites, Cyt-c, and Myo (200 ng/L for each, 50 L) were chosen as standard protein samples. In this study, we choose 10 min as digestion time in order to get the best digestion efficiency of MG@PDA-trypsin nanocomposites within the shortest time without microwave assistance. After 10-min digestion at 37⬚C, the trypsin immobilized nanocomposites were easily retrieved with a magnetic field from solution. For comparison, proteins were also treated by traditional 16-h in-solution digestion at 37⬚C. The digested peptides were analyzed by MALDI-TOF MS, and the results are shown in Fig. 3 and Supporting Information Table 1. According to the results, it is obvious that the digestion efficiency obtained by MG@PDA-trypsin nanocomposites was comparable to that obtained by traditional in-solution digestion, which was owing to the higher enzyme concentration. The sequence coverage obtained from the database of MG@PDA-trypsin nanocomposites is 62% for Cyt-c and 83% for Myo, which is similar to those of other reported www.proteomics-journal.com
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IMERs. For example, in Jiang’s work, the sequence coverage obtained from the database of trypsin-immobilized GO-CONH-Fe3 O4 nanocomposites is 60% for Cyt-c and 84% for Myo [27]. The results indicate that trypsin immobilized MG@PDA nanocomposites are beneficial to improve the digestion efficiency. The digestion efficiency of MG@PDA-trypsin nanocomposites for very small amount of protein with low concentration was investigated. For this purpose, 50 L of 1 ng/L Cyt-c was chosen as a sample. As shown in Supporing Information Fig. 3, after 10-min digestion, six peptides were matched and the corresponding sequence coverage was 39%, which demonstrated the ability of as-made nanocomposites for digestion of low abundance proteins. The non-specific adsorption of peptides on the as-made MG@PDA-trypsin nanocomposites was investigated. First, the MG@PDA-trypsin nanocomposites were employed to digest Myo. After washing with 25 mM NH4 HCO3 for three times, the nanocomposites were used for further digestion of Cyt-c. The results indicate that after digestion of Cyt-c, no peptides of Myo were obtained in the Cyt-c digests while ten tryptic peptides of Cyt-c were matched with the sequence coverage of 60%. The low peptide residue on MG@PDA-trypsin nanocomposites is in virtue of good hydrophile of PDA. The reusability and stability of MG@PDA-trypsin nanocomposites were also evaluated using standard protein Myo as the sample. After five consecutive digestions, the obtained peptides in the fifth time were almost the same as those in the first time (12 common matched peptides), which indicated that the as-made nanocomposites could be reused at least five times with the same effects of digestion. To test the stability of the trypsin immobilized materials, the as-prepared nanocomposites were stored at 4⬚C for one month. The obtained results of digestion were still the same as those obtained by freshly made nanocomposites (sequence coverage of Myo remained as 83%). The above results show the good reusability and stability of MG@PDA-trypsin nanocomposites. The reproducibility of different batches of MG@PDA nanocomposites as well as trypsin immobilization on different batches of MG@PDA nanocomposites were investigated in this study. We repeated the experiments about the reproducibility several times. And all the results are similar to each other. The sequence coverage obtained from the database of MG@PDA-trypsin nanocomposites is around 60% for Cyt-c and 80% for Myo. And the matched peptides were about 11 for Cyt-c and 14 for Myo. The results indicate that the reproducibility of MG@PDA nanocomposites is very good, which enable the pre-synthesized nanocomposites to be an ideal matrix of trypsin immobilization for digestion in complex samples. Encouraged by the high digestion efficiency and good reproducibility of MG@PDA-trypsin nanocomposites, the as-made nanocomposites were further applied in largescale proteomics research. Extracted proteins from skimmed milk were selected to evaluate the digestion capacity for C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. Venn of the overlap of identified proteins from skimmed milk between in solution and MG@PDA-trypsin digestion.
the complex real sample. After digestion at 37⬚C, the obtained peptides were analyzed by nano LC-ESIMS/MS. The Venn of overlap of identified proteins from skimmed milk between traditional in-solution digestion and MG@PDA-trypsin nanocomposites is shown in Fig. 4. As depicted in Supporing Information Table 2 and 3, 321 proteins can be identified with the as-made nanocomposites for 10-min digestion, while only 264 proteins are identified with 16-h in-solution digestion. The overlap is 123 proteins, which is not so large. We speculate the reason why the overlap of identified proteins by MG@PDA-trypsin nanocomposites and in-solution digestion is not so large is relative to the structure and the way for trypsin immobilization of MG@PDAtrypsin nanocomposites, which makes the enzyme digestion sites different from those of in-solution digestion. We chose graphene, the two-dimensional layers of sp2 -bonded carbon, as the substrate to support a high loading capability. Dopamine was employed to provide catechol and quinone functional groups present in the PDA coating, which are capable of covalent coupling to amine groups of trypsin. Thus the structure of graphene and the way for trypsin immobilization with dopamine may result in different enzyme digestion sites between two methods, which are responsible for the non-overlapping nature of in-solution and MA@PDA-trypsin nanocomposites. Although the true reason is not clear, the above results still demonstrate that the MG@PDA-trypsin nanocomposites have high digestion capacity even for complex real samples. The authentic reason is worth to studying in our next work. In summary, magnetic graphene nanocomposites coated with PDA shell were synthesized by a simple approach, trypsin was then immobilized on the surface through PDA chemistry. The as-made nanocomposites were successfully applied in the digestion of proteins. Owing to the large surface of grapheme and high hydrophilicity of PDA, the novel nanocomposites not only have high trypsin immobilization capacity (up to 0.175 mg/mg) which led to a high digestion efficiency of proteins, but also offer a simple sample preparation procedure due to the good magnetic responsiveness of magnetic microspheres. Within 10 min, the peptide sequence coverage of protein was comparable to that obtained by traditional 16-h in-solution digestion. In addition, the asmade nanocomposites exhibited good reusability, stability, www.proteomics-journal.com
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and non-specific adsorption, and were performed well in very small amounts of proteins and complex real samples. Therefore, this strategy offered a novel approach to immobilize trypsin for high digestion efficiency and indicated a great potential for high-throughput proteome profiling. This work was supported by the National Basic Research Priorities Program (2012CB910601 and 2013CB911201), the National Natural Science Foundation of China (21075022, 20875017, and 21105016), Research Fund for the Doctoral Program of Higher Education of China (20110071110007, 20100071120053), Shanghai Municipal Natural Science Foundation (11ZR1403200), and Shanghai Leading Academic Discipline Project (B109). The authors have declared no conflict of interest.
References [1] Kahn, P., From genome to proteome: looking at a cell’s proteinstitle. Science 1995, 270, 369–370. [2] Domon, B., Aebersold, R., Mass spectrometry and protein analysis. Science 2006, 312, 212–217. [3] Jang, H. B., Sung, H. W., Nho, S. W., Park, S. B. et al., Enhanced reliability of avian influenza virus (AIV) and Newcastle disease virus (NDV) identification using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Anal. Chem. 2011, 83, 1717–1725. [4] Spro, J., Sinz, A., A capillary monolithic trypsin reactor for efficient protein digestion in online and offline coupling to ESI and MALDI mass spectrometry. Anal. Chem. 2010, 82, 1434–1443. [5] Ma, J., Zhang, L., Liang, Z., Shan, Y., Zhang, Y., Immobilized enzyme reactors in proteomics. Trends Anal. Chem. 2011, 30, 691–702. [6] Wang, S., Bao, H., Zhang, L., Yuan, P., Chen, G., Infraredassisted on plate proteolysis for MALDI-TOF-MS peptide mapping. Anal. Chem. 2008, 80, 5640–5647. [7] Winter, D., Steen, H., Optimization of cell lysis and protein digestion protocols for the nalysis of Hela S3 cells by LCMS/MS. Proteomics 2011, 11, 4726–4730. [8] Wang, S., Bao, H. M., Yang, P. Y., Chen, G., Immobilization of trypsin in polyanliline-coated nano-Fe3 O4 .carbon nanotube composite for protein digestion. Anal. Chim. Acta. 2008, 612, 182–189. [9] Qiao, L., Liu, Y., Hudson, S. P., Yang, P. Y. et al., A nanoporous reactor for efficient proteolysis. Chem. Eur. J. 2008, 14, 151– 157. [10] Bornscheuer, U. T., Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. 2003, 42, 3336–3337. [11] Ma, J., Liang, Z., Qiao, X., Deng, Q. et al., Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal. Chem. 2008, 80, 2949– 2956.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2014, 14, 1457–1463 [12] Brinkworth, C. S., identification of ricin in crude and purified extracts from castor beans using on-target tryptic digestion and MALDI mass spectrometry. Anal. Chem. 2010, 82, 5246– 5252. [13] Xu, F., Wang, W., Tan, Y., Bruening, M. L., Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion. Anal. Chem. 2010, 82, 10045–10051. [14] Nagai, N., Suzuki, Y., Sekikawa, C., Nara, T. Y. et al., Fabrication of boehmite films with cage-like pores and their properties as enzyme immobilization supports. Mater. Chem. 2012, 22, 3234–3241. [15] Pierre, S. J., Thies, J. C., Dureault, A., Cameron, N. R. et al., Covalent enzyme immobilization onto photopolymerized highly porous monoliths. Adv. Mater. 2006, 18, 1822– 1826. [16] Lin, S., Yao, G., Qi, D., Li, Y. et al., Fast and efficient proteolysis by microwave-assisted protein digestion using trypsinimmobilized magnetic silica microspheres. Anal. Chem. 2008, 80, 3655–5665. [17] Liu, Z., Wang, H. S., Li, B., Liu, C. et al., Biocompatible magnetic cellulose-chitosan hybrid gel microspheres reconstituted from ionic liquids for enzyme immobilization. J. Mater. Chem. 2012, 22, 15085–15091 [18] Dulay, M. T., Baca, Q. J., Zare, R. N., Enhanced proteolytic activity of covalently bound enzymes in photopolymerized sol gel. Anal. Chem. 2005, 77, 4604–4610. [19] Pereira-Medrano, A. G., Forester, S., Fowler, G. J. S., McArthur, S. L., Wright, P. C., Rapid fabrication of glass/PDMS hybrid IMER for high throughput membrane proteomics. Lab Chip 2010, 10, 3397–3406. [20] Gao, J. H., Gu, H. W., Xu, B., Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Accounts Chem. Res. 2009, 42, 1097–1107. [21] Li, Y., Zhang, X. M., Deng, C. H., Functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis. Chem. Soc. Rev. 2013, 42, 8517–8539. [22] Qin, W. J., Song, Z. F., Fan, C., Zhang, W. J. et al., Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18 O labeling and absolute protein quantification. Anal. Chem. 2012, 84, 3138–3144. [23] Huang, J., Li, X., Zheng, Y., Zhang, Y. et al., Immobilization of penicillin G acylase on poly[(glycidyl methacrylate)co-(glycerol monomethacrylate)]-grafted magnetic microspheres. Macromol. Biosci. 2008, 8, 508–515. [24] Deng, Y. H., Deng, C. H., Qi, D. W., Liu, C. et al., Synthesis of core/shell colloidal magnetic zeolite microspheres for the immobilizaton of trypsin. Adv. Mater. 2009, 21, 1377–1382. [25] Novoselov, K. S., Geim, A. K., Morozov, S. V., Jiang, D. et al., Electric field effect in atomically thin carbon films. Science, 2004, 306, 666–669. [26] Stoller, M. D., Park, S. J., Zhu, Y. W., An, J. H., Ruoff, R. S., Graphene-based ultracapacitors. Nano Lett. 2008, 8, 3498– 3502. [27] Jiang, B., Yang, K. G., Zhao, Q., Wu, Q. et al., Hydrophilic immobilized trypsin reactor with magnetic graphene oxide
www.proteomics-journal.com
Proteomics 2014, 14, 1457–1463
1463
as support for high efficient proteome digestion. J. Chromatogr. A 2012, 1254, 8–13.
wall carbon nanohorns and their release. Carbon 2008, 46, 1792–1794.
[28] Lee, H., Dellatore, S. M., Miller, W. M., Messersmith, P. B., Mussel-inspired surface chemistry for multifunctional coatings. Science, 2007, 318, 426–430.
[32] Hong, S., Lee, J. S., Ryu, J., Lee, S. H. et al., Bio-inspired strategy for on-surface synthesis of silver nanoparticles for metal/organic hybrid nanomaterials and LDI-MS substrates. Nanotechnology 2011, 22, 494020.
[29] Lee, H., Scherer, N. F., Messersmith, P. B., Single molecule mechanics of mussel adhesion. Proc. Natl. Acad. Sci. USA 2006, 103, 12999–13003. [30] Yan, Y. H., Zheng, Z. F., Deng, C. H., Zhang, X. M, Yang, P. Y., Facile synthesis of Ti4+ -immobilized Fe3 O4 @polydopamine core-shell microspheres for highly selective enrichment of phosphopeptides. Chem. Commun. 2013, 49, 5055–5077. [31] Fei, B., Qian, B., Yang, Z., Wang, R. et al., Effect of hole size on the incorporation of C60 molecules inside single-
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[33] Lee, H., Rho, J., Messersmith, P. B., Facile conjugation of biomolecules onto surfaces via mussel adhesive protein inspired coatings. Adv. Mater. 2009, 21, 431–434. [34] Li, Y., Yan, B., Deng, C., Tang, J. et al., On-plate digeston of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis. Proteomics 2007, 7, 3661–3671.
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