Facile synthesis of Fe^III–tannic acid film-functionalized magnetic silica microspheres for the enrichment of low-abundance peptides and proteins for MALDI-TOF MS analysis

Abstract

In this work, novel Fe^III–tannic acid (TA) film-functionalized magnetic silica microspheres (designated Fe₃O₄@SiO₂@FTA) were prepared via a facile deposition of one-step assembled Fe^III–TA films on the surface of magnetic silica microspheres. The resulting Fe₃O₄@SiO₂@FTA microspheres possessed strong magnetic responsiveness and good dispersity in aqueous solutions. We introduced low concentrations of standard peptide, standard protein, peptides in BSA tryptic digests and human urine to investigate the enrichment capability of the prepared nanocomposites. The enrichment process was very fast (only 5 min) and the peptide/protein–magnetic microsphere conjugates were readily separated by an external magnet. Moreover, the obtained conjugates can be directly analyzed by MALDI-TOF MS without any elution procedure. The enrichment performance was quite satisfying, indicating Fe₃O₄@SiO₂@FTA microspheres as a universally applicable material for efficient and fast enrichment of peptides and proteins.

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1. Introduction

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), endowed with simplicity, excellent mass accuracy, high resolution and sensitivity, has become an indispensible analytical tool for proteomic research.^1,2 Despite these advantages, it is still challenging for MALDI-TOF MS to trace low-abundance peptides/proteins due to their extremely low concentrations in real biological samples. Moreover, highly abundant peptides/proteins in the sample matrices or contaminants like salts and surfactants introduced in pretreatment procedures would seriously interfere with the MS detection. Therefore, efficient enrichment of low-abundance peptides/proteins is critical prior to their MS detection.^3,4

Nanomaterials have been developed dramatically, providing us a brand new insight for addressing this issue. Notably, various zeolites nanocrystals,⁵ core–shell nanobeads,⁶ and metal–organic frameworks (MOFs)⁷ are reported to be effective adsorbents for the enrichment of peptides and proteins in proteomic researches. However, these techniques all require intensive centrifugation to separate target analytes from sample matrices, which is a time-consuming procedure that may also lead to the coprecipitation of undesirable large molecules, hence limiting their application in high throughput proteins/peptides profiling.^8–10 Over the past decades, functionalized magnetic nanomaterials have been applied in various fields such as enzyme immobilization, protein isolation and cell separation,¹¹ thanks to their strong magnetic responsiveness, fair biocompatibility and versatile functionality.^12–14 Recently, functionalized magnetic nanomaterials have also been employed as affinity probes for the efficient enrichment of peptides and proteins as they can facilitate the isolation of target analytes from sample solutions with the help of an external magnet.^15,16 Deng et al. functionalized silica-coated iron oxide (Fe₃O₄@SiO₂) with hydrophobic poly(methyl methacrylate) (PMMA) and efficiently enriched peptides and proteins through hydrophobic interactions.¹⁷ In Deng's another work, Cu²⁺ ions were immobilized on the surface of Fe₃O₄@SiO₂ by chelator iminodiacetic acid (IDA) and could successfully conjugate peptides by affinity interaction of Cu²⁺ with carboxylic and amino groups of peptides.¹⁸ These enrichment materials were functionalized through chemical binding, while physical adsorption, another kind of immobilization method, was also available for this purpose. Jiang et al. reported graphene-encapsulated silica magnetic microspheres (Fe₃O₄@SiO₂@G) via electrostatic assembly, which had a strong affinity for peptides and proteins via hydrogen bonding and electrostatic interactions, and could effectively concentrate them from sample matrices.¹⁹ However, all these materials mentioned above are a bit complicated to prepare, usually require long steps and high costs, thus blocking the possibility for wider application. Therefore, researchers are looking for alternative convenient, efficient and less complicated magnetic nanomaterials for the enrichment of peptides and proteins.

Tannic acid (TA) is a kind of polyphenolic compounds and has long been known for the interaction with peptides or proteins through hydrogen bonding and hydrophobic interactions.^20–22 Ejima and the workers reported a simple and rapid coating method using the one-step assembly of coordination complexes formed by TA and Fe^III on a range of substrates to prepare various films and particles.²³ The possible mechanism of the film formation was described as: free TA or small Fe^III–TA complexes initially adsorb onto the substrates' surface and are subsequently cross-linked by further Fe^III complexation.²³ Their brilliant research inspired us to count the possibility of employing the Fe^III–TA film, as a functional coat outside the silica shell and forming an effective absorbent towards peptides and proteins. In Ejima and coworkers' research, it only took 30 s to complete the immobilization of Fe^III–TA films on various substrates like polystyrene, gold and CaCO₃ microspheres. Besides, the raw materials of Fe^III–TA films are relatively cheap and readily available, making its universal application possible. Herein, we present a facile way to prepare Fe₃O₄@SiO₂@FTA microspheres for the first time. Then their enrichment performance towards low-abundance peptides and proteins were studied and the results were quite fascinating, indicating Fe₃O₄@SiO₂@FTA microsphere a universally applicable absorbent for efficient and fast enrichment of peptides and proteins.

2. Materials and methods

2.1 Materials and reagents

FeCl₃·6H₂O, sodium acetate, ethylene glycol, anhydrous ethanol and ammonium hydroxide were purchased form Nanjing Chemical Reagent (Nanjing, China). Tannic acid (TA) and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased from Aladdin Reagent (Shanghai, China). Tetraethylorthosilicate (TEOS) was purchased from Alfa Aesar (Tianjin, China). Angiotensin II (MW = 1046.2 Da, sequence is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) was purchased from GL Biochem (Shanghai, China). Cytochrome c (MW = 12 384 Da, from equine heart), α-cyano-4-hydroxycinnamic acid (CHCA), acetonitrile (ACN) and trifluoroacetic acid (TFA) of HPLC grade were bought from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA, fraction V) was purchased from Sangon Biotech (Shanghai, China). The protein spot was excised from a 2-DE gel developed in another work in our laboratory. Sequence Grade Modified Trypsin was from Promega (Madison, WI, USA). ZipTipC18 pipette tips were from Millipore (Billerica, MA, USA). Ultrapure water from Milli-Q system (Millipore) was used throughout. All other chemicals were of analytical grade unless otherwise noted.

2.2 Preparation of Fe₃O₄@SiO₂@FTA microspheres

Procedures for the synthesis of Fe₃O₄@SiO₂@FTA microspheres were illustrated in Scheme 1. Firstly, Fe₃O₄@SiO₂ microspheres were prepared by coating a layer of silica on Fe₃O₄ nanoparticles through a sol–gel process to prevent the nanoparticles from agglomeration and oxidation. After that, the Fe₃O₄@SiO₂ microspheres were further functionalized with Fe^III–TA films though one-step assembly (see experimental details in ESI†).

Scheme 1 Synthesis of Fe₃O₄@SiO₂@FTA microspheres.

2.3 Characterization of materials

TEM images were taken on a JEOL JEM-200CX (Tokyo, Japan) microscope operated at 200 kV. Samples were first dispersed in ethanol and then collected using carbon-film-covered copper grids for analysis. SEM images and energy dispersive X-ray (EDX) spectra were captured on a Hitachi S-3400N II (Tokyo, Japan) electron microscope operated at 20 kV. FT-IR spectra were collected on a Nicolet Fourier spectrophotometer using KBr pellets (Waltham, MA, USA). Magnetic hysteresis loops were measured on a Quantum Design SQUID VSM (San Diego, CA, USA) magnetometer. Zeta potentials were measured on a BIC 90Plus (New York, NY, USA).

2.4 Enrichment of peptides and proteins

Sample solutions of standard peptide, standard protein, tryptic digests of BSA, tryptic digests of protein spots from a 2-DE gel, and human urine were prepared. Adsorption capacity of Fe₃O₄@SiO₂@FTA microspheres was investigated using cytochrome c as test protein (see experimental details with Fig. S2 in ESI†).

After sample preparations, we added Fe₃O₄@SiO₂@FTA microspheres suspension (2 μL of 3.3 mg mL⁻¹) to 500 μL sample solutions mentioned above. The result mixtures were agitated for 5 min (see experiment optimization with Fig. S3 in ESI†) at room temperature. Then, with the help of a magnet, the protein/microspheres or peptide/microspheres conjugates were separated and rinsed with water three times. After that, the obtained microspheres were redispersed in 4.5 μL of a mixture of 10 mg mL⁻¹ CHCA (in 50% (v/v) ACN and 0.1% (v/v) TFA aqueous solution). Finally, 1.5 μL of above slurry was deposited on the MALDI plate, and applied for MALDI-TOF MS analysis.

2.5 MALDI-TOF MS analysis

All Samples were analyzed in positive ion mode on a 4800 Plus MALDI TOF/TOF TM Analyzer (Applied Biosystems, USA) with the Nd-YAG laser at 355 nm and an acceleration voltage of 20 kV. Except for human urine sample was performed in the linear mode, other analysis of peptides was performed in the reflector mode. Proteins were identified using MASCOT search engine (database: SwissProt; digested used: trypsin; maximum of missed cleavages: 1; mass tolerance: ±50 ppm).

3. Results and discussion

3.1 Characterization of Fe₃O₄@SiO₂@FTA microspheres

TEM measurement was used to identify the structure and morphology of the Fe₃O₄@SiO₂@FTA microspheres. As shown in Fig. 1a and b, the as-prepared Fe₃O₄@SiO₂ microspheres are nearly spherical-shaped. The mean diameter of the magnetic core was about 330 nm and the average thickness of the silica shell was about 20 nm. A representative TEM image (Fig. 1c) of Fe₃O₄@SiO₂@FTA microspheres suggests the formation of a coordination complexes layer on the surface of Fe₃O₄@SiO₂ microspheres after the simple functionalization. SEM images also indicate that the Fe₃O₄@SiO₂@FTA microspheres are endowed with narrow size distribution and good dispersity (Fig. S1†), in good agreement with the TEM results.

Fig. 1 TEM images of Fe₃O₄ (a), Fe₃O₄@SiO₂ (b) and Fe₃O₄@SiO₂@FTA microspheres (c), and the corresponding magnetic hysteresis loops (d).

The magnetic properties of the obtained materials were investigated with a vibrating sample magnetometer at room temperature. The maximum saturation magnetization values of Fe₃O₄, Fe₃O₄@SiO₂, and Fe₃O₄@SiO₂@FTA were calculated to be 71.7, 53.0, and 46.1 emu g⁻¹, respectively. As shown in Fig. 1d, the hysteresis loops of the three samples suggest that they all exhibit strong magnetic responsiveness, which are beneficial to the practical application because these materials would be easily collected when an external magnetic field applied.

The FT-IR spectra of Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂@FTA also testified the successful coating of Fe^III–TA complexes films onto the surface of Fe₃O₄@SiO₂ microspheres. In Fig. 2a, the absorption peak for Fe₃O₄@SiO₂ at around 1082 cm⁻¹ is assigned to the Si–O–Si vibration. The adsorption bands around 1626 cm⁻¹ and 3412 cm⁻¹ can be ascribed to hydroxyl groups and water absorbed on the particles. New absorption peaks are observed in the spectra of Fe₃O₄@SiO₂@FTA (Fig. 2b). The peaks at around 1614 cm⁻¹, 1487 cm⁻¹, 1440 cm⁻¹, and 1703 cm⁻¹ can be attributed to the C=C and C=O bonds from benzene rings, and ester groups of TA, respectively, which suggests the successful deposition of Fe^III–TA films on the Fe₃O₄@SiO₂ microspheres.

Fig. 2 FT-IR spectra of Fe₃O₄@SiO₂ (a) and Fe₃O₄@SiO₂@FTA microspheres (b).

Zeta potential measurement was also conducted throughout and gave the similar results. As shown in the Table S1,† formation of the Fe^III–TA films on the Fe₃O₄@SiO₂ particles shifted the surface zeta potential from −30.08 mV to −34.26 mV due to the acidic nature of TA.²³

3.2 Enrichment of low abundance proteins and peptides

In order to investigate the enrichment efficiency of Fe₃O₄@SiO₂@FTA microspheres, a standard peptide, angiotensin II (4 nM, Mr = 1046.2 Da, pI = 6.74) and a standard protein, cytochrome c (40 nM, Mr = 12 384 Da, pI = 10.0–10.5) were chosen as models. The samples were prepared in water (pH = 7.0). After concentration by agitation for only 5 min at room temperature, an external magnetic field was applied to separate the protein/microspheres or peptide/microspheres. Then the obtained conjugates were prepared for MS analysis with the matrix α-CHCA (10 mg mL⁻¹, in 50% (v/v) ACN and 0.1% (v/v) TFA aqueous solution).

Fig. 3a and d show the MALDI-TOF mass spectra of 4 nM angiotensin II before and after concentration with Fe₃O₄@SiO₂@FTA microspheres. Apparently, at a concentration as low as 4 nM, angiotensin II was hardly detectable and the signal-to-noise (S/N) ratio was only 51.70. After treatment with Fe₃O₄@SiO₂@FTA microspheres, the peak intensity of angiotensin II increased sharply with the S/N ratio of 5217.35, and the enrichment factor was more than 100, suggesting the Fe₃O₄@SiO₂@FTA microspheres a highly efficient enrichment material in peptide analysis. Commercial absorbent ZipTipC18 and Fe₃O₄@SiO₂ microspheres were also tested for comparison. As shown in Fig. 3b, after treatment with ZipTipC18 pipette tip, the S/N ratio only raised to 272.50 with an enrichment factor of around 5. Although Fe₃O₄@SiO₂ microspheres were capable of enriching angiotensin II (Fig. 3c), its enrichment factor was only a quarter of the result of Fe₃O₄@SiO₂@FTA microspheres. Thus it can be clearly seen that a very simple and superfast functionalization with Fe^III–TA films on Fe₃O₄@SiO₂ microspheres could dramatically improve the enrichment capacity. Fig. 4 shows the enrichment effects of the material towards 40 nM cytochrome c. The S/N ratio of cytochrome c increased up to 1197.03 after treatment with Fe₃O₄@SiO₂@FTA microspheres, whereas the S/N ratio was only 24.33 without any enrichment. Besides, replicate analysis of the two model samples were carried out. The obtained S/N ratios were listed in Table S2,† indicating that the novel material has a stable enrichment performance and delivers a good data reproducibility.

Fig. 3 MALDI-TOF MS spectra of angiotensin II (4 nM) without treatment (a), after treated with ZiptipC18 (b), with Fe₃O₄@SiO₂ (c) and Fe₃O₄@SiO₂@FTA microspheres (d).

Fig. 4 MALDI-TOF MS spectra of cytochrome c (40 nM) without treatment (a), after treated with ZiptipC18 (b), with Fe₃O₄@SiO₂ (c) and Fe₃O₄@SiO₂@FTA microspheres (d).

To evaluate the universality of this novel material in the enrichment of peptides, very dilute solution of tryptic digests of BSA was employed. At a very low concentration of 2.5 nM, only 4 weak peaks can be detected in the MS spectrum (Fig. 5a). After treated with Fe₃O₄@SiO₂@FTA microspheres, the number of peaks detected (marked with asterisks) increased up to 14 (Fig. 5c and Table S3†) with an overall improvement in the S/N ratio of MS signals (Table S4†). Commercial absorbent ZiptipC18 and Fe₃O₄@SiO₂ microspheres were also used to enrich the tryptic digests of BSA. Comparing to Fe₃O₄@SiO₂@FTA microspheres, fewer peaks with lower intensity were detected when the solution was treated with ZiptipC18 (Fig. 5b). Even though Fe₃O₄@SiO₂ microspheres showed some enrichment effect on standard peptides, they failed to enrich tryptic digests of BSA (Fig. 5c), which was in a good agreement with the previous report.¹⁹ Considering their relatively weaker enrichment capability, ZiptipC18 and Fe₃O₄@SiO₂ microspheres were no longer employed in further tests of complex biological samples in this work.

Fig. 5 MALDI-TOF MS spectra of BSA tryptic digests (2.5 nM) without treatment (a), after treated with ZiptipC18 (b), with Fe₃O₄@SiO₂ (c) and Fe₃O₄@SiO₂@FTA microspheres (d).

The material was also proven to be effective in real proteomic analysis. A protein spot was randomly chosen and excised from a 2-DE gel and digested with a general protocol as described in ESI (refer to Fig. S4†). In order to evaluate the enrichment efficiency of Fe₃O₄@SiO₂@FTA microspheres, the tryptic digests extracted from the protein spot were diluted 50 folds with water to form a low concentration solution of peptide mixtures. As shown in Fig. S5,† no peaks were detected in the MS spectrum of the dilute peptide mixtures solution. When performing PMF search, no peptides were matched and the protein sequence coverage ratio can't be determined, resulting in the failure to identify the protein separated from the 2-DE gel. However, after enriched with Fe₃O₄@SiO₂@FTA microspheres, both the intensity and S/N ratio of MS signals increased sharply and the protein was successfully identified with a score of 84 and a protein sequence coverage ratio of 35%. Detailed information was listed in Table S5.†

Urine, due to its non-invasive character and wide availability, has become a very important human specimen in clinical diagnosis.¹⁰ Peptidome profiling of human urine has proven to be an effective method for investigating kidney physiology and detecting novel disease-associated markers of renal and bladder diseases.²⁴ In this study, we further attempted to examine the application possibility of using Fe₃O₄@SiO₂@FTA microspheres to enrich peptides from human urine. Fig. 6a shows the MS spectrum of the prepared dilute urine solution before any enrichment. No peaks were detected because of the poor MS signal caused by the low concentration peptides in urine sample. However, after a fast enrichment with Fe₃O₄@SiO₂@FTA microspheres, several peaks of urine peptides were detected by MALDI-TOF MS (Fig. 6b) with decent intensity and S/N ratio of MS signals, which indicated that the as-prepared microspheres possess the ability of enriching low-abundance peptides as well as keeping unaffected by the negative influence of urea and other contaminants existed in complex biological samples. These characters suggested the Fe₃O₄@SiO₂@FTA microspheres a potential material in the enrichment of low-abundance peptides from real biological samples.

Fig. 6 Mass spectra for human urine peptide profiling before (a) and after treatment with Fe₃O₄@SiO₂@FTA microspheres (b).

3.3 Comparison with other previously reported materials

Detailed comparisons including S/N enrichment factor towards standard peptides/proteins, enrichment effects towards tryptic digests, and real samples with other similar materials are shown in Table S6.† From Table S6,† we can conclude that our material Fe₃O₄@SiO₂@FTA has comparable enrichment effects with those materials reported in previous works.

4. Conclusions

In summary, we prepared the novel Fe^III–TA films-functionalized magnetic silica microspheres (Fe₃O₄@SiO₂@FTA) and applied this magnetic material to the enrichment of low-abundance peptides and proteins in several samples varied from simple standard peptide/protein solution to complex biological samples like human urine. The as-synthesized microspheres exhibit excellent magnetic responsibility which facilitated the separation of peptides/protein–microspheres conjugates from the sample solution. Besides, there are several advantages of the novel nanocomposites comparing to other magnetic nanomaterials reported in previous articles. Firstly, the functionalization process is very fast, only taking 30 s to realize, which is much shorter than the time needed in the synthesis procedures of other magnetic nanomaterials. Secondly, the raw materials of the functional Fe^III–TA films are low-cost and readily available, which might help extend the enrichment application to a large-scale situation. Thirdly, the obtained peptides/proteins–microspheres conjugates after magnetic separation can be analyzed directly without any elution procedure which can reduce sample loss. And most importantly, along with all these merits, the enrichment performance of Fe₃O₄@SiO₂@FTA microspheres is also satisfying in comparison with other magnetic materials. Therefore, the “double economical” (both time and money) synthesis strategy of Fe^III–TA films-functionalized magnetic silica microspheres and the fast, efficient enrichment procedure towards low-abundance peptides and proteins opens the possibility for further application of the promising material in proteomic researches.

Acknowledgements

This work was supported by National Basic Research Program of China (973 program, 2011CB911003), National Natural Science Foundation of China (21275069), National Science Funds for Creative Research Groups (21121091), and Analysis & Test Fund of Nanjing University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra05872d

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