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Hydrophilic Polydopamine-Coated Graphene for Metal Ion Immobilization as a Novel IMAC Platform for Phosphoproteome Analysis Yinghua Yan, Zhifang Zheng, Chun-Hui Deng, Yan Li, Xiangmin Zhang, and Pengyuan Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401668e • Publication Date (Web): 13 Aug 2013 Downloaded from http://pubs.acs.org on August 26, 2013
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Hydrophilic Polydopamine-Coated Graphene for Metal Ion Immobilization as a Novel IMAC Platform for Phosphoproteome Analysis Yinghua Yan1, Zhifang Zheng1, Chunhui Deng∗,1, Yan Li*,2, Xiangmin Zhang1, Pengyuan Yang1 1
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
2
Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203, China.
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ABSTRACT: To discover trace phosphorylated proteins or peptides with great biological significance for in-depth phosphoproteome analysis, it is urgent to develop a novel technique for highly selective and effective enrichment of phosphopeptides. In this work, an IMAC (immobilized metal ion affinity chromatography) material with polydopamine coated on the surface of graphene, and functionalized with titanium ions (denotes as Ti4+-G@PD) was initially designed and synthesized. The newly prepared Ti4+-G@PD with enhanced hydrophilicity and biological compatibility was characterized using SEM, TEM, and IR, and its performance for selective and effective enrichment of phosphopeptide was evaluated with both standard peptide mixtures and human serum.
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Protein phosphorylation is one of the most important protein post-translational modifications (PTM); it plays a crucial role in eukaryotic cells involved in regulation of cell division, growth, migration, differentiation, and intercellular communication,1-3 and investigations into this process are of keen interest in the field of proteomics.4-6 Mass spectrometry (MS), because of its highthroughput, ultrahigh sensitivity, and simplicity in quantification of changes in phosphorylation states and identification of phosphorylation sites,7-9
has become the powerful tool for
determining the phosphorylation profiles of proteins in phosphoproteome research. However, the identification and characterization of phosphoproteins remain a challenge due to their low abundance and low ionization efficiency.10 Thus, selective enrichment of phosphoproteins or phosphopeptides from highly complicated mixtures is vital for MS-based phosphoproteome analysis. To date, various materials and techniques have recently been introduced for the selective enrichment of phosphopeptides. Immobilized metal ion affinity chromatography (IMAC), which relies on the affinity of the phosphate groups to metal ions, is the most widely used technique to capture the phosphopeptides out of the pool of predominantly non-phosphopeptides, in which the metal ions are immobilized to different adsorbents via acidic chelating ligands of iminodiacetic acid (IDA), or nitrilotriacetic acid (NTA).11-18 However, the bound metal ions were easily lost during sample loading and washing caused by the relatively weaker interaction (each metal ion only coordinates one IDA or NTA ligand). To solve this problem, Zou et al. recently introduced new ligand of phosphate group to immobilize Ti4+ or Zr4+ for phosphoproteome research.19, 20 Although IMAC technique has been highly developed, it still has some drawbacks. First, the surface area of the absorbent material is small; second, Modification of phosphate group on the absorbent is very complicated and time-consuming. To discover trace phosphorylated proteins or
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peptides with great biological significance, it is urgent to develop a novel IMAC technique to highly selective and sensitive enrichment of phosphopeptides to go through in-depth phosphoproteome analysis. Graphene has attracted considerable interest and has become the focus of much research since its discovery in 2004.21 It possesses exceptional properties such as high specific surface area (2630 m2/g), high thermal conductivity (5000 W/m K)22 and biocompatibility.23 These unique and intriguing features make this highly versatile carbon material promising in many potential applications such as transparent conducting films, sensors, bioanalysis and so on.24-26 Recently, as an excellent support, graphene decorated with metal nanoparticles, metal oxide nanospheres, and polymers has been reported.27-29 Designed and synthesis of graphene hybrid materials with high surface area for proteomics research have been become the research hot point.30-32 Recently, it has been proved that dopamine, commonly known as a hormone and neurotransmitter,33,
34
contributes to the high sticking (and cross-linking) ability of marine
adhesives. It can polymerize into a unique hydrophilic polydopamine that can adhere to a variety of substrates.35-38 The polydopamine can be readily generated in a mild condition via the oxidative self-polymerization of the dopamine in a basic solution, and it has an excellent environmental stability, good biocompatibility and especially the excellent dispersibility in water.39 Moreover, the metal ions such as Fe3+ and Ti4+ can be directly immobilized on the polydopamine.40 Therefore, hydrophilic polydopamine obtained by simple synthesis approach can be used as the novel ligand for metal ion immobilization for phosphoproteomics research. Recently, Ti4+-Fe3O4@polydopamine microspheres were successfully designed and synthesized for efficient and selective enrichment of phosphopeptides in biological samples.41 The aim of this work was to design and synthesize a novel IMAC material with polydopamine coated on the
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surface of graphene, and functionalized with titanium ions (denotes as Ti4+-G@PD) for phosphoproteome research. The grafting layer of the polydopamine-decoration would provide enhanced hydrophilicity and biocompatibility. On the other hand, the high specific surface area of graphene would offer higher capacity for loading dopamine and thus enhance the amounts of immobilized titanium ions which would result in a larger capacity of phosphopeptides binding. The Ti4+-G@PD with the above unique properties was anticipated to have excellent performance for the selective enrichment of phosphopeptides, which would be highly beneficial for mass spectrometric analysis.
RESULTS AND DISCUSSION Synthesis and Characterization of Ti4+-G@PD. The synthesized procedure for the Ti4+G@PD was illustrated in scheme S1a. In brief, self-assemble polymerization of dopamine was performed on graphene in basic condition, then the immobilization of Ti4+ on G@PD was performed by the chelation reaction of dopamine with Ti4+. The Ti4+-G@PD obtained by the proposed approach was characterized by SEM, TEM, and IR. Fig. 1a displayed the SEM image of the Ti4+-G@PD, showing a uniform shape and a considerable rough surface after coating with PD, which suggested the successful synthesis of surface-immobilized graphene nanomaterials with high surface coverage. TEM image of Ti4+G@PD was displayed in Fig. 1b, from which it could be clearly observed that, after modification with PD, the plane structure of graphene was well retained due to the good chemical and physical stability of graphene.
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(b)
Figure 1. Images of SEM (a) and TEM (b) of Ti4+-G@PDA. To test whether the dopamine had a significant effect on the immobilized capacity of titanium ions, the amount of immobilized titanium ions on the Ti4+-G@PD was measured by the energydispersive X-ray analysis (EDX) (Fig. S1 and Table S1). As depicted in Fig. S1, it can be seen that the immobilized amount of titanium on G@PD was very large, indicating the contribution of the high density of hydroxyl groups on dopamine for immobilization of titanium ions. Also, thanks to the large surface area of graphene (2630 m2/g), it could be easily estimated that the surface area of G@PD should be much larger than Fe3O4@PD, and thus lead to a higher loading capacity of titanium ions. The assumption was proved by the EDX results. As demonstrated in Table S1, it can be seen that the titanium weight (%) of Ti4+-G@PD was about two times than that of Ti4+-Fe3O4@PD. Fourier transform infrared spectroscopy (FTIR) was employed to characterize Ti4+-G@PD. After coating with PD, many new peaks can be seen in Ti4+-G@PD (Fig. S2, curve a), the absorption band at 1622 cm-1 was attributed to N-H stretching and 1494 cm-1 and 1442 cm-1 were assigned to benzene ring C-C vibration. The C-O stretching of phenolic O-H was located at 1280 cm-1, implying that graphene had been successfully modified by PD polymers via the simple oxidative polymerization method. Due to the hydrophobic property, the
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graphene cannot be dispersed in water. They would quickly precipitate out of the aqueous dispersion even treated with ultrasonication (Fig. S3a); while after coated with polydopamine, the material exhibited excellent water dispersibility without any sedimentation due to the presence of numerous hydrophilic moieties, such as hydroxyl and amine groups (Fig. S3b). The improved water dispersibility of Ti4+-G@PD made it an ideal IMAC material in MALDI-TOF mass spectrum analysis. In summary, the process of Ti4+ immobilization on G@PD is very facile and due to the unique properties of the obtained Ti4+-G@PD, it possesses the following merits. First, the thicker grafting layer of the polydopamine-decoration provided the enhanced hydrophilicity and biocompatibility. Second, high specific surface area of graphene enabled higher loading of dopamine and thus enhanced the amounts of immobilized titanium ions which was obviously beneficial for binding more phosphopeptides. The Ti4+-G@PD with the above properties was anticipated to have excellent performance for the selective enrichment of phosphopeptides. Investigation of the Performance of Ti4+-G@PD for Phosphopeptide Enrichment. A typical procedure to enrich phosphopeptide from a tryptic digest of proteins or peptides was displayed in Scheme S1b. Namely, the tryptic digest of proteins or peptides was mixed and incubated with Ti4+-G@PD for 0.5 h, and then the IMAC materials were washed. The captured phosphopeptides on the Ti4+-G@PD material were eluted and deposited on the MALDI target for MALDI-TOF MS analysis. In order to investigate the enrichment capacity of the Ti4+-G@PD toward phosphopeptides, a tryptic digest from 1×10-6 M of β-casein was incubated in the presence of the material. After separation of the material and thorough washing, the captured phosphopeptides were eluted for MALDI-TOF MS analysis. For comparison, before enrichment, the β-casein digest was also analyzed by MS, with the results presented in Fig. S4a. Only some
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nonphosphopeptides could be detected but no phosphopeptide peaks. The same amount of βcasein was also incubated with the Ti4+-G@PD, with the MALDI-TOF MS spectrum illustrated in Fig. S4b. The signals for the phosphopeptides (marked with asterisk) significantly increased and dominated the spectrum and non-phosphopeptides peaks disappeared, indicating a high enrichment capacity of the Ti4+-G@PD. The selectivity of Ti4+-G@PD for phosphopeptide enrichment was investigated using the tryptic digests of β-casein and BSA mixture as the sample. As shown in Fig. 2a, when the molar ratio of β-casein and BSA was 1:1000, without enrichment, no phosphopeptide peak was observed, while non-phosphopeptide peaks were of high intensity. However, after enrichment by Ti4+-G@PD, the S/N ratio of phosphopeptides was significantly improved, leading to the identification of five phosphopeptides with clear background (Fig. 2b). The result indicated that Ti4+-G@PD could be used in the enrichment of phosphopeptides, even at low concentration.
(a)
(b)
Figure 2. MALDI mass spectra of peptides derived from a peptide mixture of β-casein and BSA at a molar ratio of 1: 1000 (5×10-11 mol of β-casein and 5×10-8 mol of BSA): (a) without enrichment (b) enriched by Ti4+-G@PD. The phosphopeptides were marked with the asterisks. After enrichment, an aqueous solution of NH4OH (5 µL, 0.4 M) was added to elute the captured phosphopeptides, and 0.5µL of elute was spotted onto MALDI plate and analyzed by MS.
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Another obvious advantage of Ti4+-G@PD was the high sensitivity towards phosphopeptides. As shown in Fig. 3a, phosphopeptides in 10 fmol of β-casein digests could be easily detected by MALDI-TOF MS after enrichment by Ti4+-G@PD. Two phosphopeptides could still be identified when the amount of β-casein digests was as low as 1 fmol (Fig. 3b), which was much lower than many previous reports.41, 42 The improved enrichment capability and lower detection limit may be due to that the Ti4+-G@PD material possessed good biocompatibility and especially the excellent dispersibility in water, which could effectively reduce the loss of phosphopeptides through the strong hydrophilic interaction between the groups of dopamine and phosphopeptides. The result further demonstrated that materials with good biocompatibility and excellent water dispersibility were vital for phosphoproteome analysis. Control experiment was conducted by using Ti4+-Fe3O4@PD and Ti4+-G@PD to capture phosphopeptides from modern β-casein, βcasein and BSA mixtures. The experimental results validated that the enrichment capability and selectivity of Ti4+-G@PD were better than Ti4+-Fe3O4@PD, and the detection limit of Ti4+G@PD was lower than Ti4+-Fe3O4@PD.
(a)
(b)
Figure 3. MALDI mass spectra of phosphopeptides enriched by Ti4+-G@PD, with the tryptic digests of β-casein amount as (a) 10 fmol and (b) 1 fmol. The phosphopeptides were marked with the asterisks. After enrichment by Ti4+-G@PD, an aqueous solution of NH4OH (5 µL, 0.4
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M) was added to elute the captured phosphopeptides, and 0.5µL of elute was spotted onto MALDI plate and analyzed by MS. In summary, compared to Ti4+-Fe3O4@PD which was prepared in our previous work,41 Ti4+G@PD material could enrich phosphopeptides when the molar ratio of phosphopeptodes/nonphosphopeptides was high up to 1:1000, and the detection limit was 1 fmol. While for Ti4+Fe3O4@PD material, it could selectively enrich phosphopeptides when the molar ratio of phosphopeptodes/non-phosphopeptides was 1:500, and the detection limit was 2 fmol. According to the above results, it can be concluded that the newly prepared Ti4+-G@PD exhibited better selectivity and sensitivity, in other words, the replacement of Fe3O4 by graphene did enhance the phosphopeptide enrichment performance. Whether Ti4+-G@PD can be recycled and reused for capturing phosphopeptides was also tested. Ti4+-G@PD was recycled by washing with the buffer solution. The regenerated Ti4+G@PD was reused to capture phosphopeptides from β-casein digest for five consecutive times. As shown in Fig. S5, the MS spectrum of phosphopeptides after enrichment for five times was similar to that for the first time, confirming that Ti4+-G@PD can be recycled and reused for capturing phosphopeptides. With the purpose of examining the effectiveness and selectivity of the Ti4+-G@PD in the enrichment of phosphopeptides from a complex sample, human serum which contains endogenous phosphopeptides was selected as the real sample. As shown in Fig. 4, the peak of nine phosphopeptides could be clearly detected after enrichment. In comparison, no phosphopeptides were observed before enrichment and the peaks of nonphosphopeptides dominated the spectra. To the best of our knowledge, this is the highest selectivity hitherto
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observered.43-45 The results suggested that Ti4+-G@PD were capable of selective trapping phosphopeptides from naturally obtained complex sample.
(a)
(b)
Figure 4. MALDI mass spectra of peptides derived from human serum (a) without enrichment and (b) enriched by Ti4+-G@PD. The phosphopeptides were marked with the asterisks.
CONCLUSIONS In conclusion, a novel IMAC material, the Ti4+-G@PD was successfully synthesized by introducing titanium ions on the polydopamine coating of graphene for the enrichment of phosphopeptides with improved specificity, sensitivity and recovery, compared with other previous reported materials.46,
47
Ti4+-G@PD
exhibited the enhanced hydrophilicity and
biological compatibility and showed high-performance in the selective and effective enrichment of phosphopeptides from peptide mixtures, this work was expected to open up a new possibility to design a more efficient and sensitive tool for phosphoproteome analysis.
ASSOCIATED CONTENT Supporting Information. This material includes experimental methods, additional data of Ti4+-G@PD characterization and its performance in phosphopeptides enrichment. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Authors *Prof. C. H. Deng, E-mail: chdeng@fudan.edu.cn, Fax: +86-21-65641740. *Dr. Y. Li, E-mail: yanli@fudan.edu.cn Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the National Basic Research Priorities Program (2012CB910601), the National Natural Science Foundation of China (21075022, 20875017, 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).
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