Polydopamine Grafted Porous Graphene as Biocompatible

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Polydopamine Grafted Porous Graphene as Biocompatible Nanoreactor for Efficient Identification of Membrane Proteins Xiaoni Fang, Jingjing Zhao, Kun Zhang, Pengyuan Yang, Liang Qiao, and Baohong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00407 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

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ACS Applied Materials & Interfaces

Polydopamine Grafted Porous Graphene as Biocompatible Nanoreactor for Efficient Identification of Membrane Proteins Xiaoni Fang, Jingjing Zhao, Kun Zhang, Pengyuan Yang, Liang Qiao,* Baohong Liu* Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China.

ABSTRACT: Functional nanomaterials, used as nanoreactors, have shown great advantages in a variety of applications in biomedical fields. Herein, we designed a novel nanoreactor system towards the application in membrane proteomics by using polydopamine coated nanoporous graphene foams (NGFs-PD) prepared by a facile in-situ oxidative polymerization. Taking advantage of the unique 3D structure and surface functionalization, NGFs-PD can quickly adsorb large amount of hydrophobic membrane proteins dissolved in sodium dodecyl sulfonate (SDS)/methanol and hydrophilic trypsin in aqueous solution, and then confine the proteolysis in the nanoscale domains to fasten the reaction rate. Therefore, the current nanoreactor system combines the multi-functions of highly efficient solubilization, immobilization, and proteolysis of membrane proteins. With the nanoreactor, digestion of standard membrane proteins can be finished in 10 min. 893 membrane proteins were identified from human glioma cells (U251). All these superiorities indicate that the biocompatible NGFs-PD nanoreactor system is of great promise to facilitate high-throughput membrane proteomic analysis.

ACS Paragon Plus Environment


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KEYWORDS: nanoreactor, porous graphene, membrane protein, proteolysis, mass

spectrometry

1. INTRODUCTION In recent years, nanostructured materials with controlled morphologies, tailored structures, and designed functions have drawn great attention for a range of applications in biomedical area such as protein purification, targeted drug delivery, cell separation, and medical diagnosis.1-8 Based on the nanomaterials, a variety of nanoreactors have been developed and gained immense interest due to their properties, such as ultra-efficient reaction and potential application in sensitive and high-throughput bioanalysis.9-14 Substantial progress has been made in the application of nanoreactors to protein science, where the nanoreactors are used for protein extraction, enrichment and confined enzymatic reaction, which are all vital procedures in mass spectrometry (MS)-based proteomics.15-20 Despite the tremendous recent success, most of the developed nanoreactors for proteomic research are mainly focused on global proteins. For specific proteins with diverse properties, e.g. hydrophobic membrane proteins, dedicated nanoreactors should be further developed in the view of target-directed discovery of functional proteins. Membrane proteins are of crucial importance in a variety of physiological and pathological processes, including molecular transport, cell communication, and signal transduction.21-23 Comprehensive and detailed analysis of membrane proteins in a proteome-wide scale is of great values for understanding these procedures. Currently, MS based techniques have become the most important and powerful tools for


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identification, quantification, and structure characterization of membrane proteins as well as their posttranslational modification.23-25 Nevertheless, because of their inherently low abundance and highly hydrophobic nature, membrane proteins are very difficult to be efficiently extracted and digested by enzyme to peptides for subsequent MS analysis. Thus, an efficient solubilization, separation, enrichment, and digestion procedure prior to MS analysis is urgently demanded. To address the aforementioned issues, a number of techniques have been developed to facilitate the MS based analysis of membrane proteins. MS compatible additives are often required to first fully solubilize membrane proteins regardless of their interference to enzyme activity, separation efficiency of liquid chromatography (LC), and MS signals.26-30 After efficient solubilisation by highly concentrated formic acid (FA), acid-compatible cyanogen bromide (CNBr) or pepsin is commonly employed to digest membrane proteins instead of trypsin, followed with MS analysis.28,31,32 Although the developed techniques did improve the number of identified membrane proteins, the shortage of degradation specificities results in the dramatic increase of possible digested peptides, which leads to time-consuming data analysis and high false discovery rate, ultimately to inefficient identification of membrane proteins. Recently,

solvent-free

matrix

assisted

laser

desorption

ionization

mass

spectrometry (MALDI−MS), two-phase digestion, and on-membrane digestion approaches have drawn great attention in the analysis of membrane proteins and enabled efficient membrane proteins identification.33,34 Nevertheless, the methods always turn out to be time-consuming and involve cumbersome procedures. Therefore,



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there is still an urgent demand to develop efficient, simple and cost-effective strategies for membrane proteins identification. To date, there are few reported techniques to integrate the enhanced solubilization, enrichment, and digestion of membrane proteins in one system, which will be very important for high-throughput membrane proteomics.35-36 It is then desirable to design a novel nanoreactor system, which can realize all the functions. Graphene with 3-D structure has been reported as a good candidate for nanoreactor.36 It possesses exceptional properties, such as high specific surface area, interconnected porous structure, biocompatible microenvironment, and highly hydrophobicity, due to the combination of a porous structure with the excellent intrinsic properties of graphene.37, 38 These unique and intriguing features make the 3-D graphene promising in enriching hydrophobic membrane proteins within its nanopores to accelerate enzymatic reaction rate and reduce digestion time. However, because the bare 3-D graphene does not have desired interactions with enzyme, i.e. trypsin, the enriched membrane proteins cannot be efficiently digested. Polymeric coating is a widely used method to tailor surface property. Among the developed polymers, polydopamine (PD) has turned out to be an important surface modification reagent, which can be readily generated via the self-polymerization of dopamine in alkaline buffer.39 It has high cross-linking ability, extraordinary biocompatibility, excellent environmental stability, and dispersibility in water.40-42 Moreover, endothelial cells and proteins can be directly immobilized on PD via the formation of a covalent bond.41, 42 Therefore, by combining the unique functions of


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3-D graphene and PD, an easy-to-prepare nanoreactor towards membrane proteomic research can be expected. Herein, polydopamine coated nanoporous graphene foams (NGFs-PD) were synthesized by a simple postgrafting approach. We have found that the NGFs-PD showed super good water dispersibility and fast extraction kinetics to both trypsin and hydrophobic membrane proteins. Membrane proteins dissolved in sodium dodecyl sulfonate (SDS) containing methanol were effectively extracted by the NGFs-PD. Trypsin was subsequently added and also extracted by the material, so that both enzyme and substrate could be confined in the inner pores to reach a high local concentration and a nanoconfinement effect for enhanced enzymatic reaction rate. With the NGFs-PD as nanoreactor, standard membrane proteins can be digested in 10 minutes for MS characterization. When a complex proteomic sample (human glioma cells) is considered, 893 membrane proteins were identified. All the results demonstrated that such a biocompatible nanoreactor system could offer a promising tool for the characterization of membrane protein with high efficiency and throughput.

Scheme 1. Schematic illustration of the synthesis of NGFs-PD materials.



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2. RESULTS AND DISCUSSION 2.1 Synthesis and characterization of the NGFs-PD materials. The synthesis method of the polydopamine coated nanoporous graphene foams (NGFs-PD) is illustrated in Scheme 1. Firstly, NGFs were fabricated according to the hard template method.37 TEM image of NGFs clearly shows the highly porous structure with spherical pores (Figure S1a, Supporting information), which is also observed by AFM (Figure S1b, Supporting information). The polydopamine-functionalized porous graphene (NGFs-PD) was synthesized by an in-situ polymerization method. The morphology and characteristic of the NGFs-PD materials were studied by various methods. As shown in Figure 1a&b, the porous structure of NGFs can be well preserved after surface coating by PD. N2 adsorption/desorption isotherms measurement further validates the porous property of NGFs-PD (Figure 1c). The BET surface area and pore size of NGFs-PD are 417.2 m2/g and 13.1 nm, respectively. To confirm the chemical modification of NGFs with PD, FT-IR spectroscopy was employed. As shown in Figure 1d, the absorbance peaks of NGFs-PD at 3343 cm−1 and 1616 cm-1 are ascribed to the O–H stretching vibration and the aromatic N-H stretching vibration, respectively. The peak at 1500 cm-1 can be assigned to benzene ring C-C vibration. The adsorption bands between 1400 cm-1 to 600 cm-1 contain -CH2 bending vibration (1344 cm-1), C-O-H asymmetric bending vibration (1285 cm-1), C-O asymmetric bending vibration (1261 cm-1), C-N stretching vibration (1147 cm-1) and Ar-H bending vibration assigned to 1, 2, 4-substitued aromatic compounds (876 and 790 cm-1). These results indicate that NGFs have been successfully modified


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by PD polymers via the facile in-situ oxidative polymerization method.

Figure 1. (a) TEM images of NGFs-PD. (b) 3D AFM view of NGFs-PD obtained with NanoScope Analysis v1.40 software. (c) Pore size distribution of NGFs-PD and corresponding N2 adsorption/desorption isotherms (inset). (d) FT-IR spectra of NGFs-PD.

The modification of NGFs with PD was further validated by Raman spectra (Figure 2a). In the spectrum of NGFs, the three strong characteristic peaks at 1336, 1567, and 2678 cm−1 are assigned to the D, G and 2D mod of NGFs. The D mode, a disorder-activated Raman mode, is associated with the defects in the sp3 carbon, graphite sheet, or other impurities. The G mode is related to the movement of two neighboring carbon atoms in the opposite direction of a graphitic sheet.43 For pure PD, the broad peaks at 1350 and 1600 cm−1 are attributed to the stretching and



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deformation of catechols.44 In the spectrum of NGFs-PD, all the characteristic peaks remain at their respective positions although the peaks became slightly broadened. These results indicate that the modification of NGFs with PD polymers through the mild solution-phase polymerization is a nondestructive approach for engineering the surface property of NGFs. After the modification of hydrophilic PD, the NGFs-PD exhibit excellent water dispersibility without any sedimentation (Figure 2b). We also found that the NGFs-PD materials could still be well dispersed even after one month of storage (Figure S2, Supporting information). Apart from the improved hydrophilic property and stability, the PD coating also provides abundant ligand sites for the enrichment of membrane proteins via covalent interactions,41 as illustrated in Scheme 2.

Figure 2. (a) Raman spectra of NGFs-PD and (b) comparison of the solubility in water for NGFs (left, 1 mg/mL) and NGFs-PD (right, 1 mg/mL).

To demonstrate the feasibility of using NGFs-PD materials as an integrated nanoreactor system in the characterization of membrane proteins (Scheme 2), their extraction ability to membrane proteins as well as trypsin was quantitatively characterized. Bacteriorhodopsin (BR), an integral membrane protein was used as a model hydrophobic membrane protein substrate. Compared with other solvents,


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methanol containing 2% SDS shows the highest dissolving ability to BR (Figure 3a). The NGFs-PD exhibited highly fast adsorption kinetics and large loading capacity to the well-dissolved BR. Without the PD coating, although the adsorption is still fast, the maximum loading capacity is limited. As shown in Figure 3b, more than 96.3 % of the well-dissolved BR (1 mg/mL) can be enriched by NGFs-PD nanoreactor within 5 min, the maximum adsorption amount is 238 mg BR (g NGFs-PD) −1. However, only less than 60% BR can be adsorbed into pure NGFs materials within the same time, demonstrating the key role of PD in the enrichment of membrane proteins through the specific covalent interactions. In the case of trypsin, the maximum immobilization capacity is 392 mg (g NGFs-PD) −1. Similarly, most trypsin can be adsorbed within 5 min (Figure 3c). In contrast, the maximum loading capacity to trypsin by bare NGFs is only 56 mg (g NGFs) −1.

Scheme 2. Schematic illustration of the NGFs-PD nanoreactor system assisted efficient enrichment and digestion of well-dispersed membrane proteins. It should be



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noted that the NGFs-PD is actually much larger than proteins and enzyme. The well-dissolved membrane proteins should be extracted either in the nanopores or on the surface of NGFs-PD during the centrifugation and washing steps.

Therefore, with the NGFs-PD, well-dispersed substrate and enzyme can be rapidly captured and concentrated by the nanoporous material to realize fast kinetics of enzymatic reactions due to the nanoconfinement and enrichment effect in nanoreactors.17 In addition, the hydrophilic PD coating ensures that the nanoreactor can be well-dispersed in an aqueous solution containing 25mM ammonium bicarbonate (ABC), where trypsin can reach the highest enzymatic activity. As illustrated in Figure 3d, the trypsin activity in 25 mM ABC is much higher than that in the usually used solvent (60% methanol, 100% methanol, 2% SDS in methanol) for the solubilization/tryptic digestion of membrane proteins.


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Figure 3. (a) Solubility of membrane protein (BR) in different solvent. (b) Extraction of membrane protein (BR, in methanol containing 2% SDS with the initial concentration of 1 mg/mL) by the materials (0.5 mg/mL) as a function of incubation time. (c) Adsorption of trypsin in ABC aqueous solution (25 mM, pH 8.0) with an initial concentration of 1 mg/mL into the materials (0.5 mg/mL) as a function of incubation time. (d) Trypsin activity in different solvent (25 m M ABC (k1), 60% methanol (k2), 100% methanol (k3), and methanol containing 2% SDS (k4)). The substrate was BAEE with an initial concentration of 0.8 mg/mL. The absorbance (Abs) corresponds to BA at 253 nm, which was generated from BAEE by trypsin-catalyzed hydrolysis. The concentration of enzyme was 0.5 mg/mL.



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2.2 Application of the NGFs-PD nanoreactor system in the analysis of membrane proteins. All the aforementioned results indicate that the NGFs-PD nanoreactor system can be used to host membrane proteins and enzyme and thereby to assist the analysis of membrane proteins. We used firstly BR as a standard protein to demonstrate this concept. The membrane protein was firstly dissolved in methanol containing 2% SDS, then extracted by the NGFs-PD nanoreactor, followed with centrifugation and redispersion in 25 mM ABC aqueous solution containing trypsin for efficient in-nanopore digestion. After proteolysis, peptides are eluted by 0.1% TFA/50% ACN aqueous solution (v/v) for MS analysis. The elution procedure is indispensable. As shown in Figure S3 (Supporting information), only a small peak with low intensity and high background corresponding to peptide of BR can be detected in the supernatant of the NGFs-PD nanoreactor system-assisted digestion before elution. Figure 4a-d display the peptide mass fingerprinting (PMF) spectra obtained by traditional overnight digestion and the nanoreactor-assisted proteolysis. For direct comparison, the same protein concentration (100 ng/µL) and enzyme/protein ratio (1:10 w/w) were used for all experiments. As shown in Figure 4a&b, much more peptides originated from BR were observed by the NGFs-PD nanoreactor system-assisted digestion (14 peptides) compared to the 10 min in-solution digestion (2 peptides). Even after 12 h of in-solution digestion, the number of match peptides (9 peptides, Figure 4c) was still not comparable to that obtained from the 10 min NGFs-PD

nanoreactor

system-assisted

proteolysis.

To


be

mentioned,

the

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solubilisation of BR by 25 mM ABC aqueous solution was accomplished with the assistance of sonication. For unmodified NGFs nanoreactor-assisted proteolysis system, there was no peptide from BR detected after 10 min of digestion (Figure 4d), probably because of the bad dispersibility of NGFs in 25 mM ABC aqueous solution. Stability of NGFs-PD material is very important for membrane proteomic research. As shown in Figure S4 (Supporting information), by using NGFs-PD materials dispersed in water after 1 month to assist the Aproteolysis of BR, a similar peptide mass fingerprinting (PMF) result could be achieved.

Figure 4. (a) Mass spectrum obtained after 10 min NGFs-PD nanoreactor-assisted digestion of BR (100 ng/µL, in methanol containing 2% SDS). The digestion was performed in 25 mM ABC aqueous solution. Mass spectra obtained after (b) 10 min and (c) 12 h in-solution digestion of BR (100 ng/µL) in 25 mM ABC aqueous solution. (d) Mass spectrum obtained after 10 min NGFs nanoreactor-assisted digestion of BR (100 ng/µL, in methanol containing 2% SDS). The digestion was performed in 25 mM



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ABC aqueous solution. (e) Mass spectrum obtained after 10 min NGFs-PD nanoreactor-assisted BR (100 ng/µL, in 100% methanol) digestion. The digestion was performed in 25 mM ABC aqueous solution. (f) Mass spectrum obtained after 10 min NGFs-PD nanoreactor-assisted digestion of BR (100 ng/µL, in methanol containing 2% SDS). The digestion was performed in 60% methanol. All the identified peptides are labeled with corresponding m/z. The enzyme to substrate ratio was always 1:10 (g:g).

We have further compared the influence of different solvent for membrane protein solubilisation and tryptic digestion. With the NGFs-PD as nanoreactor, when 100% methanol without SDS was used to prepare BR solution (100 ng/µL) for NGFs-PD extraction, followed with 10 min digestion in 25 mM ABC aqueous solution, 10 matched peptides were observed (Figure 4e), which is slightly worse than the result in Figure 4a, indicating that methanol with 2% SDS is a better solvent to prepare membrane protein solution. With the solvent for membrane protein solubilisation and 60% methanol in water for tryptic digestion, which is commonly used for the tryptic digestion of non-water soluble protein, 10 peptides were identified (Figure 4f), demonstrating that still the 25 mM ABC aqueous solution maintains the best enzymatic activity of trypsin, in accordance with Figure 3d. It’s worth noting that all the identified peptides by other methods were also identified by the integrated NGFs-PD nanoreactor system with optimal solvent conditions (Figure 4a). The good performance of the integrated NGFs-PD nanoreactor system is presumably attributed to the following reasons: (1) the unique 3-D structure provides favorable supports to the digestion by adsorbing both protein and trypsin into the


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inner-pores and confining the following reaction in the nanoscale domain; (2) the coated PD offers enough ligand sites for the enrichment of well dissolved proteins through covalent interaction, which can not only greatly improve the enrichment capacity but also avoid the interference of nonspecific adsorption of SDS; (3) the hydrophilic PD coating improves the dispersibility of the nanoreactor in alkaline aqueous solution that is optimal for trypsin activity. In summary, NGFs-PD can serve as an ideal medium to link the good solubility of membrane proteins in SDS containing methanol and the optimal activity of trypsin in 25 mM ABC aqueous solution.

Figure 5. Mass spectra of BR at different concentrations (a) 50, (b) 20, (c) 10, and (d) 1 ng/µL after 10 min proteolysis by the NGFs-PD nanoreactor system. All the identified peptides are labeled with corresponding m/z.

With the optimal NGFs-PD assisted digestion of membrane proteins, we have further tested the method in the analysis of low concentration samples. Indeed, because of the low abundance of membrane proteins in cells, significant progress for



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the detection of low concentration membrane proteins is highly demanded. When the protein concentrations were lowered to 50, 20, 10, and 1 ng/µL, 10, 8, 6 and 3 tryptic peptides were identified, respectively, after the NGFs-PD assisted digestion (Figure 5). In contrast, no peptides could be identified from 1 ng/µL BR after proteolysis by other protocols as mentioned in Figure 4. Such results further demonstrate that the NGFs-PD nanoreactor system is highly advantageous for the efficient solubilization, enrichment, and tryptic digestion of membrane proteins.

2.3 Application of the NGFs-PD nanoreactor system to the analysis of membrane proteins from real-case biological samples. Encouraged by the unique features of the developed NGFs-PD Nanoreactor System, proteins extracted from U251 cells were prepared in methanol containing 2% SDS, digested by the nanoreactor protocol, and then analyzed by nano-RPLC-ESI/MS/MS for identification. Figure 6a displays the results after database searching. A total of 2,156 peptides and 625 proteins were identified. In comparison, the in-solution digestion approaches (see detail in the supporting information) just allowed identification of 234 proteins by the same LC-MS/MS method, among which only 29 proteins were missed by the NGFs-PD nanoreactor system. The transmembrane hidden Markov model (TMHMM) analysis shows that 218 of the 625 proteins are integral membrane proteins. The detailed information of the identified membrane proteins is listed in Table S1 (Supporting Information). The distribution of the number of transmembrane domains (TMDs) of the identified


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membrane proteins was also studied. As shown in Figure S5 (Supporting information), the proteins with one TMD contributed more than half of all the identified membrane proteins. Proteins with 6 to 8 TMDs and 11 to 13 TMDs were exclusively identified by the NGFs-PD nanoreactor system. The grand average of hydrophobicity (GRAVY) values of the identified membrane proteins were further studied. Figure 6b shows the distribution of GRAVY and isoelectric point (PI) of all the identified membrane proteins by the NGFs-PD nanoreactor system. The GRAVY values of identified membrane proteins range from -1.1 to 1.1, and hydrophobic membrane proteins (GRAVY≤0) are more notable than the hydrophilic ones (GRAVY>0). The PI of the identified membrane proteins vary in the range of 4 to 12, indicating that both acidic and basic membrane proteins can be enriched by the NGFs-PD nanoreactor.

Figure 6. (a) Distribution and overlap of the identified proteins. The proteins were digested by the NGFs-PD nanoreactor system and in-solution approach before LC-MS/MS analysis. (b) Distribution of PI and GRAVY values of the identified membrane proteins from U251 cells by the NGFs-PD nanoreactor system assisted proteolysis coupled with nano-RPLC-ESI-MS/MS analysis.



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To achieve the global membrane proteins analysis of U251 cell, 2D-nano LC-ESI-MS/MS analysis was carried out for the digests of membrane proteins from the NGFs-PD nanoreactor system assisted proteolysis and the in-solution digestion. In triplicate runs, 4284 proteins and 28922 peptides were successfully identified after proteolysis by the nanoreactor, among which 893 proteins possess at least one predicted TMDs (Supplementary file, “Excel 1.xls”). However, only 430 peptides corresponded to 332 proteins were identified using the in-solution based method. The distributions of the TMDs and GRAVY of the identified membrane proteins by both methods were further compared (Figure 7), which showed similar trends as the 1D nano-RPLC-ESI-MS/MS analysis. Compared to the traditional in-solution method, the proposed NGFs-PD nanoreactor system exhibits remarkable enhancement in the sample preparation of membrane proteins. In addition, the unique 3-D structure of NGFs-PD is favorable to the identification of membrane proteins with different molecular weight. As shown in Figure S6 (Supporting information), the molecular weight of protein up to 318.38 kDa can be identified successfully. The majority of the identified peptides have length between 7 and 20 amino acid residues (Figure S7, Supporting information), indicating that the method do not bias toward the analysis of different lengths of peptides and exhibits a typical bottom-up proteomic analysis result.


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Figure 7. Comparison of the distribution of (a) TMDs and (b) GRAVY of the identified membrane proteins from U251 cell by the NGFs-PD nanoreactor system assisted

and

traditional

in-solution

digestion

method

coupled

with

2D

nano-LC-MS/MS analysis.

3.0 CONCLUSIONS In summary, NGFs-PD material was successfully synthesized by introducing versatile dopamine modified 3-D graphene and served as an integrated nanoreactor system for the MS based analysis of membrane proteins. Taking advantages of the



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unique features of graphene foams and dopamine, the rational designed material can realize the multifunctions including protein solubilization, enrichment, and highly efficient proteolysis. As a result, the NGFs-PD nanoreactor system provides a simple but integrated protocol for membrane protein analysis that can be universally applied to high-throughput membrane proteomics. Such a breakthrough will also lead to new advances in other bio-application where enzymatic reactions are involved.

ASSOCIATED CONTENT Supporting Information The detailed information of experimental methods, mass spectrum obtained from the supernatant, identified membrane proteins by nano-RPLC-ESI-MS/MS, and comparison of the distribution of TMDs of the identified membrane proteins from U251 cell after proteolysis by the NGFs-PD nanoreactor system and traditional in-solution method coupled with nano-RPLC-MS/MS analysis. This material is available free of charge via internet at http://pubs.a.org/.

AUTHOR INFORMATION Corresponding Author *E-mail: *(B. L.) [email protected]. Fax:(+86) 21-6564-1740 *(L. Q.) [email protected]. Fax:(+86) 21-6564-1740

Author Contributions X. Fang, L. Qiao, P. Yang and B. Liu conceived research; X. Fang, J. Zhao, and K.


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Zhang performed the research; X. Fang, L. Qiao, J. Zhao, and K. Zhang analyzed the data; X. Fang, L. Qiao, and B. Liu wrote the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by NSFC (21175028, 21375022 and 21105014) and L. Qiao acknowledges financial support from Fudan University, the “1000 Youth Talents” Plan of China.

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