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NiCoMnO4: A Bifunctional Affinity Probe for His-tagged Protein Purification and Phosphorylation Sites Recognition Xiaoyue Qi, Long Chen, Chaoqun Zhang, Xinyuan Xu, Yiding Zhang, Yu Bai, and Huwei Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04280 • Publication Date (Web): 06 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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ACS Applied Materials & Interfaces

NiCoMnO4: A Bifunctional Affinity Probe for Histagged Protein Purification and Phosphorylation Sites Recognition Xiaoyue Qi a, Long Chen a, Chaoqun Zhang b, Xinyuan Xu a, Yiding Zhang a, Yu Bai a* and Huwei Liu a*

a

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry

and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing100871, China. Tel: +86-10-62758198, E-mail: [email protected], [email protected]. b

Beijing Nuclear Magnetic Resonance Center, College of Life Science, Peking University,

Beijing 100871, China.

KEYWORDS: affinity probes, His-tagged proteins purification, phosphopeptides enrichment, metal oxide, buffer modulation

ACS Paragon Plus Environment

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ABSTRACT: A bifunctional affinity probe NiCoMnO4 was designed and prepared with controllable morphology and size using facile methods. It was observed that the probe could be applied in His-tagged proteins purification and phosphopeptides enrichment simply through the buffer modulation. NiCoMnO4 particles showed satisfactory cycling performance for His-tagged proteins purification and broad pH-tolerance of loading buffer for phosphopeptides affinity. Therefore, a high-throughput, cost-effective and efficient protein/peptide purification method was developed within 10 min based on the novel bifunctional affinity probe.

1. INTRODUCTION To make a comprehensive understanding of the structure and functions of proteins, even the interplay of proteins and signalling pathways in cells, recombinant protein plays an indispensable key role.1-5 Owing to the minimal impact on the expression and folding of the target protein, polyhistidine tag (His-tag) is the most common and widely used in the above mentioned fields.

3-5

The purification of the His-tagged proteins can be realized by the

immobilized metal ion affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC), in which different metal ions and nanomaterials have been reported, such as Cu2+, Ni2+, Co2+ and Zn2+,3 Ni/NiO core/shell nanoparticles, Fe3O4/AuANTA-Co2+ nanoparticles, nickel silicate nanospheres, etc.6-8 Even though Nickelnitrilotriacetic acid (Ni-NTA) is so far the most common and the best commercialized product for His-tagged proteins purification, it still remains time-consuming, high-cost and leaking of nickel ions. Several groups synthesized Ni2+ or Co2+ immobilized magnetic nanoparticles as an affinity adsorbent for His-tagged protein purification.

7, 9-10

Inevitably,

the leaking of metal ions during loading and eluting process led to poor recyclability.

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Otherwise, the regeneration process was required to maintain the binding capacity of the material, which was cumbersome.9 Wang and co-workers developed Fe3O4@NiSiO3 for separation of His-tagged proteins. 11 The reusability of Fe3O4@NiSiO3 was satisfactory after 5 cycles. However, the incubation time and release time was 30 min, which was timeconsuming. Plus, long-time treatment increased the risk of protein degradation when employed in biological samples. Thus, a high-throughput, rapid, low-cost and efficient purification method should be developed based on novel affinity probes. NiCoMnO4 is negative temperature coefficient (NTC) thermistor with controllable morphology and facility of synthesis, integrating the properties of different transit metal ions.12 Furthermore, it was reported that Co2+ presented better affinity than Ni2+ in His-tagged protein purification.7, 13 Reversible

protein

phosphorylation,

as

a

vitally

significant

post-translational

modification, plays a critical and pleiotropic role of cell physiology regulation.14-15 Phosphorylation of specific protein, especially kinases, could initiate necroptosis,16 apoptosis,17 autophagy18-19 and cancer.20 Therefore, detecting the phosphorylation sites is valuable to provide primary evidence for identifying the signaling cascades, discovering the mechanism of enzymatic activity regulation and figuring out protein-protein interactions.14 Nevertheless, detecting the phosphorylation particularly of targeted protein remains challenging due to their low levels or technical limitations.21 Over the last decade, IMAC2224

and MOAC25-28 have become the most popular approaches for phosphorylated

protein/peptides enrichment. In addition to the single metal oxide, multivariate transition metal oxides integrating various elements’ properties have been reported for affinity and


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enrichment.29 Hence, NiCoMnO4 was supposed to be a bifunctional affinity probe for Histagged protein affinity and phosphorylation sites recognition.

2. MATERIALS AND METHODS

2.1 Chemicals and Materials. Nickel sulfate (NiSO4·6H2O), cobalt sulfate (CoSO4·7H2O),

manganese sulfate (MnSO4·H2O) were purchased from Xilong Chemical Co., Ltd (Shantou, Guangdong, China). Sodium carbonate (Na2CO3) and ammonium hydroxide (NH3·H2O) were obtained from Beijing Chemical Company (Beijing, China). β-casein, α-casein, trypsin, 2,5dihydroxybenzoic acid (2,5-DHB), acetonitrile (ACN) were supplied by Sigma-Aldrich. Phosphate buffered saline (PBS) was from Beijing Solarbio Science & Technology Co., Ltd. Trifluoroacetic acid (TFA) was bought from J&K Technologies Inc. Ammonium bicarbonate (NH4HCO3) was purchased from Fluka. Purified water was provided by Hangzhou Wahaha Group. (Hangzhou, Zhejiang, China). Nonfat milk was produced by Yili Industrial Group Co., Ltd. Ni-NTA agarose was obtained from Qiagen Co., Ltd. (Germany). Human serum was provided by the local hospital. All reagents employed were of analytical grade and used as supplied without further purification.

2.2 Synthesis and characterization of NiCoMnO4 particles. In a typical synthesis, aqueous

solutions (500 mL) containing NiSO4·6H2O (10 mmol), CoSO4·7H2O (10 mmol) and MnSO4·H2O (10 mmol) in a round bottom flask was prepared. 200 mL Na2CO3 (27.5 µg/mL) solution was added drop wise (4 mL/min) to the mixture as the precipitating agent at room temperature with magnetic stirring at the speed of 800 r/min. After reaction, the precipitates were collected, washed with H2O for several times, dried in oven at 70 ºC overnight, and then ground carefully in an agate mortar. The precipitates were further calcined in a muffle furnace in the air


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at 800 ºC for 15 h with the heating rate of 2 ºC/min, and subsequently cooled down naturally to room temperature. The final product was characterized by scanning electron microscopy (SEM, Hitachi S4800, 5kV), X-ray diffraction (XRD, Rigaku Dmax-2400, Cu Kα radiation, 40 kV, 100 mA) and X-ray photoelectron spectroscopy (XPS, Kratos, Al Kα radiation). For quality control from batch to batch, structure and morphology were two main factors that were taken into account. Thus, intensity and position of peaks in the XRD pattern, as well as SEM images were analysized carefully to get detailed composition of the phase and information of morphology.

2.3 Expression of His-tagged YFP. Fluorescent protein His-tagged YFP (cpVenus) was expressed in E.coli BL21 (DE3) cells with a C-terminal 6*histidine tag and purified with GE HisTrap column following routine procedures. Protein concentration was determined by absorbance at 280 nm with a molar absorbance constant of 26740 M-1CM-1. Protein sequence: GMHEEEFQFLRCQQCQAEAKCPKLLPCLHTLCSGCLEASGMQCPICQAPWPLGADTPAL ELMDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAA GITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQK NGIKANFKIRHNIELEHHHHHH. The molecular weight is 35005.6Da.

2.4 Purification of His-tagged YFP using NiCoMnO4. The obtained His-tagged YFP was diluted 1000 times in PBS before use. 200 µg NiCoMnO4 particles were added to the diluted His-tagged YFP solution (20 µg/mL, 500 µL), incubated at room temperature for 5 min with shaking. After carefully washed by PBS (containing 10mM imidazole) 2 times, the His-tagged YFP bound NiCoMnO4 particles were collected by centrifugation and then eluted with 1M


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imidazole (500 µL). After carefully washed, the retrieved NiCoMnO4 was obtained for recycle use.

2.5 Purification of His-tagged YFP using commercial Ni-NTA agarose. According to the handbook for purification of His-tagged proteins using Ni-NTA agarose, add 20 µL of a 50% slurry of Ni-NTA resin to 500 µL diluted His-tagged YFP solution (20 µg/mL). His-tagged YFP was diluted by lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The mixture was mixed gently for 30 min. After centrifugation for 10 s at 1000×g to pellet the resin, the supernatant was removed. Then the resin was washed by 100 µL wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) twice and eluted 3 times with 20 µL elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Then the eluted solution was diluted to 500 µL for subsequent detection. After careful washing, the retrieved Ni-NTA agarose was obtained for recycle use.

2.6 His-tagged GFP affinity using NiCoMnO4 particles in cell lysate. GFP was cloned to pET28a vector with restriction sites Nde I and BamH I. The recombinant plasmid with an N terminal His tag was transformed into E. coli BL21 (DE3) for protein expression. Protein sequence: MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDA TYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQE RTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNE KRDHMVLLEFVTAAGITLGMDELYKSGLRSRAQASGSEFELRRQACGRTRAPPPPPLRS GC. The molecular weight is 32015.7Da. 10 mL of Luria-Bertani (LB) medium (10 g Bacto-


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Tryptone, 5 g Bacto-yeast extract and 10 g NaCl in 1 L H2O) containing 50 ug/mL Kanamycin was inoculated with a single colony and incubated overnight at 37 °C with shaking at 195 rpm. The overnight culture was diluted in 250 mL of LB medium with antibiotics. Cells were cultivated at 37 °C with shaking until they reached an optical density of 0.8 at 600 nm and were then transferred to 25°C and induced with 0.1 g/L isopropyl-β-D-thiogalactopyranoside (IPTG) for additional 8-10 h. Finally, cells were harvested by centrifugation at 3500 g for 20 minutes and resuspended in 25 mL phosphate-buffered saline (PBS). After disrupting the cells by sonication for 20 min, soluble fractions were obtained by centrifugation at 4 °C with 12,000 rpm for 20 min. The solution was then diluted 10 times in PBS before use.

The diluted cell lysate (200 µL) were incubated with NiCoMnO4 particles (200 µg) at room temperature for 5 min with shaking. After carefully washed by PBS (containing 10mM imidazole) for several times, the His-tagged GFP bound NiCoMnO4 particles were collected by centrifugation and then eluted with 1M imidazole (50µL). The trapped His-tagged GFP (10µL) was detected using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, BioRad, USA), with the preconcentration voltage of 80 V and the separation voltage of 100 V. The obtained gel was stained with Coomassie blue. For western blotting, the gels were transferred onto polyvinylidene fluoride membranes (Millipore Co., Ltd, Billerica, MA, USA) that was then incubated with diluted anti-Histidine antibody (Boster Biological Technology Co., Ltd, Wuhan, Hubei, China) overnight at 4 °C. After carefully washing with 0.1% Tween 20 (TBST), the membranes were incubated with mouse secondary antibodies (Boster Biological Technology Co., Ltd, Wuhan, Hubei, China) for 2 h at room temperature. Immunodetection was performed using an enhanced ECL detection kit (Biotopped Life Science, Beijing, China).


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2.7 Preparation of standard protein digests. β-casein or α-casein was firstly dissolved in NH4HCO3 solution (50mM) with the addition of trypsin at a 1:40 (w/w) enzyme-to-protein ratio and sequently digested at 37 °C for 20 h. 30 µL nonfat milk was mixed with 970 µL NH4HCO3 solution (50mM), and supernatant was got after centrifugation at the speed of 14,000 rpm for 25 min afterwards. The mixture was denatured at 100 °C for 5 min and then incubated at 37 °C for 16 h with 30 µL trypsin solution (1mg/mL). 2.8 Enrichment of phosphopeptides using NiCoMnO4 particles. Typically, the tryptic digested samples were diluted to the appointed concentration, and added with 200 µg NiCoMnO4 particles in the loading buffer containing 0.1% TFA (ACN: H2O=1:1) for 5 min with shaking. The phosphopeptides bound materials were separated with supernatant removed after centrifugation. NiCoMnO4 particles were washed by the loading buffer for several times and eluted with ammonium hydroxide solution (10%) afterwards for MALDI-ToF-MS analysis. For phosphopeptides enrichment from human serum samples, the serum (10 µL) was diluted with loading buffer (ACN: H2O=1:1, containing 0.1%TFA, 90 µL) and then incubated with NiCoMnO4 particles for 5 min at room temperature. After 3 times washing with loading buffer (100 µL), the bound phosphopeptides was eluted with elution buffer (10% NH3·H2O, 10 µL). The mixture was centrifuged at 10000 rpm for 3 min, and the supernatant was collected for further MALDI-ToF-MS analysis. The enrichment step was the same when the composition and pH of loading buffer changed. 2.9 Treatment of NiCoMnO4 particles with different buffer. To evaluate whether NiCoMnO4 particles can survive in the loading buffer and eluting buffer, 10 mg particles were immersed into the buffer listed in Table S4 (25 mL) for 8 hours. The particles were collected after centrifugation and dried in the oven at 100 °C overnight. Subsequently, the powder was


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weighed again and then analyzed by X-ray diffraction (XRD, Rigaku Dmax-2400, Cu Kα radiation, 40 kV, 100 mA) and inductively coupled plasma-atomic emission spectrometry (ICPAES, Leeman Profile Spec). 3. RESULTS AND DISCUSSION 3.1 Synthesis and characterization of NiCoMnO4 particles. Ren and co-workers reported the synthesis of NiCoMnO4 spinel which was a kind of ternary metal oxide for the first time in 2009.30 Afterwards, Li et al. synthesized NiCoMnO4 using co-precipitation method.31-32 Furthermore, elemental analysis of NiCoMnO4 particles was achieved by EDS spectra, indicating the average ratio of four main elements was approximately 4:1:1:1 with cubic spineltype structure. 31-32 The NiCoMnO4 particles were synthesized using the co-precipitation together with high temperature solid state reaction following previous reports,31-33 which avoided cumbersome synthesis method and low yield and provided perfect repeatability and low cost for potential commercial use. To ensure good crystallinity and morphology of the material, the coprecipitation rate and sintering temperature should be precisely controlled. The corresponding morphology was identified by scanning electron microscopy (SEM, Fig.1 (a), Fig.1 (b)) which revealed that the as-synthesized material was uniform spherical particles composed of smaller nanoparticles with the average size of less than 100 nm. The diameter of spherical particles was ~2µm. NiCoMnO4 showed formation of the composite oxide with a spinel-type cubic structure (XRD, Fig.1 (c)). The splits in peaks indicated a secondary phase of NiO with rock salt structure, that was observed at 2θ= 37.1°, 43.2°, 62.7° and 75.3°. It was consistent with the reported results.31 The two phases of NiCoMnO4 were labeled separately in Fig. 1(c). Fig. 1(d), Fig. 1(e) and Fig. 1(f) were X-ray photoelectron spectroscopy (XPS). Four peaks were observed in both Ni 2p and Co 2p spectra. Peaks of Ni 2p3/2 and Ni 2p1/2 were located at the binding energy of


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854.9 eV and 872.5 eV accompanied with satellite peaks, demonstrating the existence of Ni2+. Fig. 1(e) showed two strong peaks of Co 2p3/2 at 780.5 eV and Co 2p1/2 at 796.0 eV, together with two weak satellite peaks, derived from Co3+ and Co2+. Two sharp peaks of Mn 2p3/2 at 642.3 eV and Mn 2p1/2 at 653.7 eV suggested the existing of Mn4+ and Mn3+. The results manifested that NiCoMnO4 was mixed-valence where Ni ions were in 2+ oxidation state, whereas Mn and Co ions were in 3+/4+ and 2+/3+, respectively.

Figure 1. Characterization of NiCoMnO4 particles: (a)-(b) SEM micrograph of NiCoMnO4; (c) XRD pattern of NiCoMnO4; XPS spectra of NiCoMnO4, (d) Ni 2p, (e) Co 2p and (f) Mn 2p.


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3.2 Purification of His-tagged proteins using NiCoMnO4. Ni2+ was widely used for Histagged proteins purification and Ni-based oxide could also be employed to achieve this purpose. 6

Herein, we proposed that the as-synthesized material might be able to anchor His-tagged

protein from complicate matrix with their special composition and morphology. To facilitate the characterization of the experiments, we first took a His-tagged yellow fluorescent protein (Histagged YFP) as an example. As illustrated in Fig. 2(a), NiCoMnO4 was incubated with Histagged YFP dissolved in PBS solution (20 µg/mL) for 5 min at room temperature. Then the Histagged YFP bound material was collected after centrifugation at 10,000 rmp. Confocal fluorescence microscopy (laser excitation at 488 nm) was used to characterize the binding (Fig. 2(b)). Presumably, His-tagged YFP interacted with NiCoMnO4 through chelation between metal ions and histidine.6-8, 34 The merged image illustrated that the captured YFP distributed mainly on the surface of NiCoMnO4 particles. In order to make it clearer, a 3D image (Fig. S1) was obtained to illustrate the distribution of captured YFP, confirming that YFP was absorbed mainly on the surface of NiCoMnO4 spherical particles. After eluted with 1M imidazole, no obvious fluorescent signal was observed under the same condition (Fig. 2(c)). The recovery of 86% was obtained from the comparison of the fluorescence intensity of the His-tagged YFP solution before and after elution, which was higher than previous NiO adsorbent (~70%).6,35 Meanwhile, the intensity of the supernatant after extraction decreased to below the detection limit. The results confirmed NiCoMnO4 material exhibited high binding efficiency to His-tagged YFP and easy elution using imidazole solution. The calculated affinity capacity of His-tagged YFP purification was 43 µg/mg. The retrieved materials could be reused for at least 8 times with higher than 60% purification capacity of His-tagged YFP (Fig. S2). While the affinity capacity of Ni-NTA declined sharply to 52% for the second use and then gradually decreased to 37% after


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five cycling (Fig. S3, Fig. S4 (a), Fig. S5). Accordingly, the fluorescent emission intensity in the supernatant gradually increased (Fig. S4 (b)). In Ni-NTA systems, NTA was used as the chelating ligand to immobilize Ni2+ via the chelation of the metal ions to the carboxylic groups on NTA. However, the relatively weaker interaction resulted in easy loss of the bound Ni2+ in the process of sample loading and washing.

36-38

So the reuse performance of Ni-NTA was not

satisfactory. Moreover, it was reported that Co2+ presented better affinity than Ni2+ in His-tagged protein purification.

7, 13

Additionally, Wegner and Spatz reported that Co3+, as a d6 ion, was

more thermodynamically stable than Ni2+ as an inert mediator ion between NTA and His-tagged proteins, with significantly higher formation constants than Ni2+ complexes under similar coordination environments. 39 XPS results showed that NiCoMnO4 was mixed-valence where Co ions were in 2+/3+. Based on these two advantages, the reuse performance of NiCoMnO4 was better than Ni-NTA.


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Figure 2. (a) Schematic diagram of his-tagged YFP enrichment; confocal laser microscopy image obtained from His-tagged YFP protein bound NiCoMnO4 (b) before elution, and (c) after elution with 1M imidazole solution (The image in the left was the dark-field image and the image in the middle labeled by ‘TD’ represented the bright-field image for which the light source was mercury lamp. The third one was the merged image of the dark-field image and bright-field one.); (d) fluorescence spectra from the solutions of His-tagged YFP before and after treatment with NiCoMnO4.


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Figure 3. (a) Confocal laser microscopy image and (b) fluorescence spectra obtained from NiCoMnO4 incubated with the mixture of His-tagged YFP protein and IgG labeled with Cy3. The images in the upper left corner and upper right corner represented two separate channels (Fig. 3(a)). The image labeled by TD was the bright-field image. The last image was the merged image of the dark-field images and brightfield one.


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To inspect the specificity and selectivity of NiCoMnO4 as the His-tagged protein affinity probe, normal mouse IgG labeled by red-emitting Cy3 was used in the control experiment. After the incubation of the mixture containing both Cy3 labeled mouse IgG and His-tagged YFP at the same concentration (20 µg/mL) with NiCoMnO4 particles for 5 min, the material was removed by centrifugation. As shown in Fig. 3(b), most of mouse IgG labeled by Cy3 was remained in the supernatant since no obvious fluorescent signal was observed in the eluent compared with the initial intensity, which was further corroborated by confocal microscopy image. Under the simultaneous irritation of 488nm (for His-tagged YFP) and 543nm (for IgG labeled by Cy3), the intensity of fluorescent signal derived from YFP was much stronger than the emission of IgG labeled by Cy3 on the surface of NiCoMnO4 particles (Fig. 3(a)). Logically, an intense red signal was observed in the supernatant after incubation with subsequently NiCoMnO4 moved (Fig. S6), which was consistent with the fluorescence spectra (Fig. 3(b)). The specificity of the NiCoMnO4 particles surpassed NiO probably because that Co2+ presented better affinity than Ni2+ in Histagged protein purification to reduce nonspecific proteins binding.7, 13Although the recovery of His-tagged YFP was decreased to 63%, indicating that the total adsorption was less, the affinity probe showed good specificity and selectivity to some extent. To demonstrate the practical utility of NiCoMnO4, the material was incubated with E. coli cell lysate containing His-tagged GFP following the above mentioned experimental procedure. SDSPAGE analysis (Fig. 4, Lane 1-4) offered strong evidence to prove that NiCoMnO4 was a powerful affinity probe for His-tagged proteins with outstanding practical value due to the high affinity of M2+ toward histidine. Lane 2 is the total lysate. After incubation with the NiCoMnO4 particles, the material was washed carefully with PBS buffer containing 10mM imidazole. Lane 3 is the passthrough solution of the last-time washing, which showed that there were almost no


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non-specific proteins, as well as the target protein. The intense band on Lane 4 indicated the NiCoMnO4 possessed excellent His-tagged protein binding properties with negligible nonspecific adsorption. The results of Western Blot using anti-His antibody (Fig. 4, Lane 5-8) showed the same results and the mass of NiCoMnO4 material was optimized to be 200 µg. 5 min was enough for NiCoMnO4 particles to absorb the target proteins. Compared with reported materials,9,11 NiCoMnO4 as adsorbent for His-tagged proteins purification was efficient with good selectivity and recyclability.

Figure 4. SDS-PAGE and Western Blot analysis of purified recombinant proteins from cell lysate. Lane 1 molecular weight marker; Lane 2 E. coli lysate; Lane 3 the fractions washed off last time from NiCoMnO4 using washing buffer; Lane 4 after treating protein-bound NiCoMnO4 particles with 1M imidazole solution; Lane 5-8 the fractions washed off from different mass of NiCoMnO4 for His-tagged GFP affinity in cell lysate, 50 µg, 100 µg, 200 µg, 500 µg, respectively.

3.3 Enrichment of phosphopeptides using NiCoMnO4. With successful application to fish His-tagged proteins, the bifunctional affinity probe was expected to enrich phosphopeptides. According to enriching procedures reported in our previous work,29, 40-41 the obtained material was employed for phosphopeptides enrichment in a batch mode.41 It was noteworthy that the incubation time and eluting time decreased to 5 min, demonstrating NiCoMnO4 is quite a


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powerful affinity probe towards phosphopeptides. MALDI-ToF mass spectra showed the results of tryptic digested β-casein, α-casein and nonfat milk after enrichment under the optimized condition (Fig. 5), nevertheless it was difficult for phosphopeptides detection before enrichment (Fig. S7, Fig. S8 and Fig. S9). As it was shown in Fig. 5(a), when the concentration of β-casein was 2×10-7 M, the enrichment was efficient and three phosphopeptides (m/z 2061, 2557 and 3123) were detected without nonspecific absorption. When β-casein digest was diluted to 2.0 × 10−10 M (Fig. 5d), the enrichment could also be achieved under such low concentration. For tryptic digested α-casein and nonfat milk samples, which constituted a more complex system, the obtained affinity probe could enrich 14 and 12 phosphopeptides, respectively. The sequences of detected phosphopeptides were listed in Table S2. It demonstrated that NiCoMnO4 is promising in actual applications for recognizing the phosphorylation sites due to the efficient enriching process. We further evaluated the specificity towards phosphopeptides enrichment using complex samples with the mass ratio of BSA: β-casein systematically increasing from 100:1 to 1000:1 (Fig. 5(e)), while the concentration of β-casein was 2.0 × 10−7 M. Though the mixture contained an overwhelming amount of BSA, the phosphopeptides of β-casein could still be anchored to the NiCoMnO4 material without obvious peaks of BSA in the mass spectra, indicating NiCoMnO4 showed preference for phosphopeptides rather than non-phosphorylated peptides. The endogenous low-abundant phosphopeptides in human serum play an important role in regulatory mechanisms.

42-43

As shown in Fig. S15, four endogenous phosphopeptides were

observed after enrichment and detailed information about the detected phosphopeptides from human serum was listed in Table S3. The results suggested that NiCoMnO4 particles were capable of selectively trapping phosphopeptides from naturally obtained complex samples. Furthermore, NiCoMnO4 showed a broad pH adaptability towards the loading buffer compared


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with the previous materials we reported29, 41 which may benefit from the multi component of the material (Fig. S12 and Fig. S13). Different from the traditional materials for phosphopeptides enrichment which could only be used in acidic solution, the pH range of loading buffer for NiCoMnO4 was from 1.35 to neutral solution (ACN: H2O=1:1). The composition of different loading buffer and zeta potential was listed in Table S1. NiCoMnO4 revealed negligible difference of performance in acidic buffer when enriching tryptic digested β-casein.


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Figure 5. Phosphopeptides enrichment properties of NiCoMnO4: MALDI-ToF MS spectra of tryptic digested (a) 2×10-7 M β-casein, (b) 2×10-7 M α-casein, (c) nonfat milk, (d) 2×10-10 M β-casein after enrichment by NiCoMnO4; (e) the tryptic digests from the mixture of BSA and β-casein (100:1, 500:1, 1000 : 1 n/n) after NiCoMnO4 treatment. (“β”, “α” represent phosphopeptides and “#” represents phosphopeptides after loss of HPO3.)

In traditional enrichment method, high selectivity was ensured by maintaining the carboxyl groups of non-phosphorylated peptides in protonated state to reduce the interferences of peptides with carboxyl groups.44 TFA was often used for pH adjustment.26 The optimal concentration of TFA in the loading buffer was quite different depending on the material used for phosphopeptides capture.29, 45 However, the high concentration of TFA suppressed the dissociation of

phosphate

groups, hindering

the enrichment of

phophopeptides.45 To investigate whether the binding selectivity was strong enough for phosphopeptides under neutral condition, a mixture of BSA and β-casein was employed. The loading buffer was ACN: H2O=1:1 solution without TFA. When the molar ratio of BSA and β-casein systemically increased from 50 to 500 as shown in Fig. S16, NiCoMnO4 could capture phosphopeptides of β-casein among whelming amount of non-phosphorylated peptides. Furthermore, the enrichment of phosphopeptides in diluted human serum in neutral buffer (ACN: H2O=1:1) could also be achieved using NiCoMnO4 particles as shown in Fig. S17. Four phosphopeptides were observed in the MS spectrum, which was the same as that in acid loading buffer. The principle of phosphopeptides affinity is based on coordination and electrostatic interactions between positively charged metal ions and negatively charged phosphate moieties of phosphopeptides.

44, 46-47

The zeta potential varied with pH and was positive at acidic pH,


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facilitating the phosphopeptides enrichment. Obviously, the zeta potential declined with the increase of pH. However, the metal ions were still positively charged in weakly acidic buffer, even at neutral pH, which probably benefited from the properties of multi-metal oxide particles. So the positively charged metal ions could interact with negatively charged phosphate groups. Furthermore, we speculate that the highly selective capture of phosphopeptides could mainly attribute to multicomponent of metal ions with mixed-valence. Each metal could coordinate to more than one phosphate group at the surface octahedral sites and vice versa.26, 38 The chelating ability of carboxyl groups to metal ions of NiCoMnO4 particles during enrichment was inferior to that of phosphate groups even in the neutral buffer. Therefore, the relative weaker interaction between metal ions and carboxylic peptides prevented the adsorption of carboxylic peptides, improving the selectivity of phosphopeptides even at neutral pH, which further proved that the NiCoMnO4 could be employed as adsorbent for phosphopeptides with high selectivity. Thus, it processed practical value under more flexible buffer control for various real samples. 3.4 Stablity of NiCoMnO4 particles. Material stability is quite an important factor to evaluate its practicability. It was reported that the chemical component and structure of NiCoMnO4 powder was quite stable as catalyst.32 Typically, the buffer used for His-tagged protein purification and phosphopeptides enrichment is listed in the Table S4. No obvious mass loss was observed after treatment with different buffer. Fig. S18 showed the content of three metal elements. No obvious loss of metal ions was observed, which demonstrated that the chemical properties of NiCoMnO4 was stable in corresponding solution. Plus, XPD patterns (Fig. S19) showed that there was almost no change in the peak intensity and peak position of the NiCoMnO4 particles treated with different buffer, indicating the structure was stable. So the


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results confirmed that NiCoMnO4 particles have valuable practicability in His-tagged protein purification and phosphopeptides enrichment with high chemical and structural stability. 4. CONCLUSIONS In summary, a bifunctional affinity probe NiCoMnO4 was designed and successfully synthesized for efficient His-tagged protein affinity and phosphopeptides enrichment. Compared with commercial Ni-NTA, NiCoMnO4 is cost efficient and presents high specificity and efficiency. Based on NiCoMnO4 particles, a high-throughput and efficient purification method was developed and the purification process could be finished within 10 min. In addition, the NiCoMnO4 particles can be reused for at least 8 times with 60% purifying capacity. As an affinity probe of phosphopeptides, it showed broader pH adaptability towards the loading buffer compared with our previous work, leading to flexible practicability for complex real samples. Therefore, it was a versatile material for target protein/peptide purification via buffer modulation. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: x x x x x x x. Experiment details; sequences of detected phosphopeptides; enrichment properties of NiCoMnO4 in different loading buffer; XRD patterns of NiCoMnO4 particles treated with different buffer and recycle use. Corresponding Author *E-mail: [email protected], [email protected]. Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Professor Peng Chen and Professor Bin Xia, College of Chemistry and Molecular Engineering, Peking University, for the kind help in the recombinant proteins. The research is financially supported by the National Natural Science Foundation of China (Nos. 21322505, 21575007 and 21527809). REFERENCES (1) Tao, Y.; Cheung, L. S.; Li, S.; Eom, J. S.; Chen, L. Q.; Xu, Y.; Perry, K.; Frommer, W. B.; Feng, L. Structure of a Eukaryotic SWEET Transporter in a Homotrimeric Complex. Nature 2015, 527, 259-263. (2) Doyle, L.; Hallinan, J.; Bolduc, J.; Parmeggiani, F.; Baker, D.; Stoddard, B. L.; Bradley, P. Rational Design of Alpha-Helical Tandem Repeat Proteins with Closed Architectures. Nature 2015, 528, 585-588. (3) Hengen, P. N. Purification of His-Tag Fusion Proteins from Escherichia Coli. Trends Biochem. Sci. 1995, 20, 285-286. (4) Hsu, H. M.; Chu, C. H.; Wang, Y. T.; Lee, Y.; Wei, S. Y.; Liu, H. W.; Ong, S. J.; Chen, C.; Tai, J. H. Regulation of Nuclear Translocation of the Myb1 Transcription Factor by Tvcyclophilin 1 in the Protozoan Parasite Trichomonas Vaginalis. J. Biol. Chem. 2014, 289, 19120-19136. (5) Brimacombe, C. A.; Ding, H.; Beatty, J. T. Rhodobacter Capsulatus Dpra Is Essential for Reca-Mediated Gene Transfer Agent (Rcgta) Recipient Capability Regulated by QuorumSensing and the Ctra Response Regulator. Mol. Microbiol. 2014, 92, 1260-1278. (6) Lee, I. S.; Lee, N.; Park, J.; Kim, B. H.; Yi, Y.-W.; Kim, T.; Kim, T. K.; Lee, I. H.; Paik, S. R.; Hyeon, T. Ni/NiO Core/Shell Nanoparticles for Selective Binding and Magnetic Separatioin of Histidine-Tagged Proteins. J. Am. Chem. Soc. 2006, 128, 10658-10659. (7) Zhang, L.; Zhu, X.; Jiao, D.; Sun, Y.; Sun, H. Efficient Purification of His-tagged Protein by Superparamagnetic Fe3O4/Au-ANTA-Co2+ Nanoparticles. Mater. Sci. Eng. C 2013, 33, 19891992.


22

Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(8) Wu, Y.; Chang, G.; Zhao, Y.; Zhang, Y. Hierarchical Fe3O4@NixSiOy Microspheres for Affinity Separation of His-tagged Proteins. J. Nanopart. Res. 2014, 16, 779-783. (9) Cao, J.; Zhang, X.; He, X.; Chen, L.; Zhang, Y. Facile Synthesis of A Ni(II)-Immobilized Core-Shell Magnetic Nanocomposite as an Efficient Affinity Adsorbent for the Depletion of Abundant Proteins from Bovine Blood. J. Mater. Chem. B 2013, 1, 3625-3632. (10) Lee, K. S.; Lee, I. S. Decoration of Superparamagnetic Iron Oxide Nanoparticles with Ni2+: Agent to Bind and Separate Histidine-Tagged Proteins. Chem. Commun. 2008, 709-711. (11) Wang, Y.; Wang, G.; Xiao, Y.; Yang, Y.; Tang, R. Yolk-Shell Nanostructured Fe3O4@NiSiO3 for Selective Affinity and Magnetic Separation of His-Tagged Proteins. ACS Appl. Mater. Interfaces 2014, 6, 19092-9. (12) Wang, J.; Yao, J.; Chen, Z.; Fan, Y.; Chang, A.; Jia, D. Effects of Sr-Doping on the Electrical Properties of MnCoNiO4 NTC Ceramics. J. Mater. Sci.:Mater. Electron. 2012, 24, 622-627. (13) Lopez-Gallego, F.; Yate, L. Selective Biomineralization of Co3(PO4)2-Sponges Triggered by His-Tagged Proteins: Efficient Heterogeneous Biocatalysts for Redox Processes. Chem. Commun. 2015, 51, 8753-8756. (14) Doerig, C.; Rayner, J. C.; Scherf, A.; Tobin, A. B. Post-Translational Protein Modifications in Malaria Parasites. Nat. Rev. Microbiol. 2015, 13, 160-172. (15) Wu, X.; Renuse, S.; Sahasrabuddhe, N. A.; Zahari, M. S.; Chaerkady, R.; Kim, M. S.; Nirujogi, R. S.; Mohseni, M.; Kumar, P.; Raju, R.; Zhong, J.; Yang, J.; Neiswinger, J.; Jeong, J. S.; Newman, R.; Powers, M. A.; Somani, B. L.; Gabrielson, E.; Sukumar, S.; Stearns, V.; Qian, J.; Zhu, H.; Vogelstein, B.; Park, B. H.; Pandey, A. Activation of Diverse Signalling Pathways by Oncogenic PIK3CA Mutations. Nat. Commun. 2014, 5, 4961. (16) Fuchs, Y.; Steller, H. Live to Die Another Way: Modes of Programmed Cell Death and the Signals Emanating from Dying Cells. Nat. Rev. Mol. Cell Biol. 2015, 16, 329-344. (17) Yabu, T.; Shiba, H.; Shibasaki, Y.; Nakanishi, T.; Imamura, S.; Touhata, K.; Yamashita, M. Stress-Induced Ceramide Generation and Apoptosis Via the Phosphorylation and Activation of Nsmase1 by Jnk Signaling. Cell Death Differ. 2015, 22, 258-273. (18) Gurkar, A. U.; Chu, K.; Raj, L.; Bouley, R.; Lee, S. H.; Kim, Y. B.; Dunn, S. E.; Mandinova, A.; Lee, S. W. Identification of Rock1 Kinase as a Critical Regulator of Beclin1Mediated Autophagy During Metabolic Stress. Nat. Commun. 2013, 4, 2189.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

(19) Wani, W. Y.; Boyer-Guittaut, M.; Dodson, M.; Chatham, J.; Darley-Usmar, V.; Zhang, J. Regulation of Autophagy by Protein Post-Translational Modification. Lab Invest. 2015, 95, 1425. (20) Ji, S.; Qin, Y.; Shi, S.; Liu, X.; Hu, H.; Zhou, H.; Gao, J.; Zhang, B.; Xu, W.; Liu, J.; Liang, D.; Liu, L.; Liu, C.; Long, J.; Zhou, H.; Chiao, P. J.; Xu, J.; Ni, Q.; Gao, D.; Yu, X. ERK Kinase Phosphorylates and Destabilizes the Tumor Suppressor FBW7 in Pancreatic Cancer. Cell Res. 2015, 25, 561-573. (21) Lavoie, H.; Therrien, M. Regulation of RAF Protein Kinases in ERK Signalling. Nat. Rev. Mol. Cell Biol. 2015, 16, 281-298. (22) Pan, L.; Iliuk, A.; Yu, S.; Geahlen, R. L.; Tao, W. A. Multiplexed Quantitation of Protein Expression and Phosphorylation Based on Functionalized Soluble Nanopolymers. J. Am. Chem. Soc. 2012, 134, 18201-18204. (23) Collins, B. C.; Gillet, L. C.; Rosenberger, G.; Rost, H. L.; Vichalkovski, A.; Gstaiger, M.; Aebersold, R. Quantifying Protein Interaction Dynamics by SWATH Mass Spectrometry: Application to the 14-3-3 System. Nat. Methods 2013, 10, 1246-1253. (24) Mirza, M. R.; Rainer, M.; Messner, C. B.; Guzel, Y.; Schemeth, D.; Stasyk, T.; Choudhary, M. I.; Huber, L. A.; Rode, B. M.; Bonn, G. K. A New Type of Metal Chelate Affinity Chromatography Using Trivalent Lanthanide Ions for Phosphopeptide Enrichment. Analyst 2013, 138, 2995-3004. (25) Tan, Y. J.; Sui, D.; Wang, W. H.; Kuo, M. H.; Reid, G. E.; Bruening, M. L. Phosphopeptide Enrichment with TiO2-Modified Membranes and Investigation of Tau Protein Phosphorylation. Anal. Chem. 2013, 85, 5699-5706. (26) Zhong, H.; Xiao, X.; Zheng, S.; Zhang, W.; Ding, M.; Jiang, H.; Huang, L.; Kang, J. Mass Spectrometric Analysis of Mono- and Multi-Phosphopeptides by Selective Binding with NiZnfe2O4 Magnetic Nanoparticles. Nat. Commun. 2013, 4, 1656-1662. (27) Li, W.; Deng, Q.; Fang, G.; Chen, Y.; Zhan, J.; Wang, S. Facile Synthesis of Fe3O4@TiO2–ZrO2 and Its Application in Phosphopeptide Enrichment. J. Mater. Chem. B 2013, 1, 1947-1961. (28) Wang, M.; Deng, C.; Li, Y.; Zhang, X. Magnetic Binary Metal Oxides Affinity Probe for Highly Selective Enrichment of Phosphopeptides. ACS Appl. Mater. Interfaces 2014, 6, 1177511782.


24

Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Li, L. P.; Zheng, T.; Xu, L. N.; Li, Z.; Sun, L. D.; Nie, Z. X.; Bai, Y.; Liu, H. W. SnO2ZnSn(OH)6: A Novel Binary Affinity Probe for Global Phosphopeptide Detection. Chem. Commun. 2013, 49, 1762-1764. (30) Ren, Y.; Ma, Z.; Qian, L.; Dai, S.; He, H.; Bruce, P. G. Ordered Crystalline Mesoporous Oxides as Catalysts for CO Oxidation. Catal. Lett. 2009, 131, 146-154. (31) Li, K.; Luo, X.; Lin, X.; Qi, F.; Wu, P. Novel NiCoMnO4 Thermocatalyst for LowTemperature Catalytic Degradation of Methylene Blue. J. Mol. Catal. A- Chem. 2014, 383-384, 1-9. (32) Li, K.; Zhao, J.; Zhang, Y.; Wu, P.; Zhang, Z. Near Room-Temperature Thermocatalysis: A Promising Avenue for the Degradation of Polyethylene Using NiCoMnO4 Powders. RSC Adv. 2016, 6, 11829-11839. (33) Shaikh, P. A.; Kambale, R. C.; Rao, A. V.; Kolekar, Y. D. Structural, Magnetic and Electrical Properties of Co-Ni-Mn Ferrites Synthesized by Co-Precipitation Method. J. Alloys. Compd. 2010, 492, 590-596. (34) Wu, Y.; Chang, G.; Zhao, Y.; Zhang, Y. Preparation of Hollow Nickel Silicate Nanospheres for Separation of His-Tagged Proteins. Dalton Trans. 2014, 43, 779-783. (35) Kim, J.; Piao, Y.; Lee, N.; Park, Y. I.; Lee, I. H.; Lee, J. H.; Paik, S. R.; Hyeon, T. Magnetic Nanocomposite Spheres Decorated with NiO Nanoparticles for A Magnetically Recyclable Protein Separation System. Adv. Mater. 2010, 22, 57-60. (36) Wang, Z. G.; Cheng, G.; Liu, Y. L.; Zhang, J. L.; Sun, D. H.; Ni, J. Z. Novel Core-Shell Cerium(IV)-Immobilized Magnetic Polymeric Microspheres for Selective Enrichment and Rapid Separation of Phosphopeptides. J. Colloid Interface Sci. 2014, 417, 217-226. (37) Yan, Y.; Zheng, Z.; Deng, C.; Li, Y.; Zhang, X.; Yang, P. Hydrophilic PolydopamineCoated Graphene for Metal Ion Immobilization as A Novel Immobilized Metal Ion Affinity Chromatography Platform for Phosphoproteome Analysis. Anal. Chem. 2013, 85, 8483-8487. (38) Ma, W.; Zhang, Y.; Li, L.; Zhang, Y.; Yu, M.; Guo, J.; Lu, H.; Wang, C. Ti4+Immobilized Magnetic Composite Microspheres for Highly Selective Enrichment of Phosphopeptides. Adv. Funct. Mater. 2013, 23, 107-115. (39) Wegner, S. V.; Spatz, J. P. Cobalt (III) as a Stable and Inert Mediator Ion Between NTA and His-Tagged Proteins. Angew. Chem. Int. Ed. 2013, 52, 7593-7596.


25


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Page 26 of 27

(40) Li, L. P.; Liu, J. Z.; Xu, L. N.; Li, Z.; Bai, Y.; Xiao, Y. L.; Liu, H. W. GdF3 as A Promising Phosphopeptide Affinity Probe and Dephospho-Labelling Medium: Experiments and Theoretical Explanation. Chem. Commun. 2014, 50, 11572-11575. (41) Li, L.; Chen, S.; Xu, L.; Bai, Y.; Nie, Z.; Liu, H.; Qi, L. Template-Free Synthesis of Uniform Mesoporous SnO2 Nanospheres for Efficient Phosphopeptide Enrichment. J. Mater. Chem. B 2014, 2, 1121-1124. (42) Zhang, L.; Zhao, Q.; Liang, Z.; Yang, K.; Sun, L.; Zhang, L.; Zhang, Y. Synthesis of Adenosine Functionalized Metal Immobilized Magnetic Nanoparticles for Highly Selective and Sensitive Enrichment of Phosphopeptides. Chem. Commun. 2012, 48, 6274-6276. (43) Hu, L,; Zhou, H.; Li, Y.; Sun, S.; Guo, L.; Ye, M.; Tian, X.; Gu, J.; Yang, S.; Zou, H. Profiling of Endogenous Serum Phosphorylated Peptides by Titanium (IV) Immobilized Mesoporous Silica Paticles Enrichment and MALID-ToF MS Detection. Anal. Chem. 2009, 81, 94-104. (44) Tang, L. A.; Wang, J.; Lim, T. K.; Bi, X.; Lee, W. C.; Lin, Q.; Chang, Y. T.; Lim, C. T.; Loh, K. P. High-Performance Graphene-Titania Platform for Detection of Phosphopeptides in Cancer Cells. Anal. Chem. 2012, 84, 6693-6700. (45) Shen, F.; Hu, Y.; Guan, P.; Ren, X. Ti4+-Phosphoate Functionalized Cellulose for Phosphopeptides Enrichment and Its Application in Rice Phosphoproteome Analysis. J. Chromatogr. B 2012, 902, 108-115. (46) Shen, F.; Hu, Y.; Guan, P.; Ren, X. Facile Preparation of Titanium Phosphate-Modified Chitosan for Selective Capture of Phosphopeptides. J. Sep. Sci. 2013, 36, 540-547. (47) Sun, J.; Liu, Y.; Su, Y.; Xia, W.; Yang, Y. Highly Efficient Enrichment of Phosvitin Phosphopeptides by Novel Magnetic Carboxymethyl Chitosan Nanoparticles Decorated with Fe (III) Ions. J. Chromatogr. B 2013, 915-916, 33-38.


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