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ZrO2 nanofiber as a versatile tool for protein analysis Hui Wang, Yaokai Duan, and Wenwan Zhong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09348 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 22, 2015

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ACS Applied Materials & Interfaces

ZrO2 nanofiber as a versatile tool for protein analysis Hui Wang, Yaokai Duan, Wenwan Zhong* Department of Chemistry, University of California, Riverside Keywords: zirconia, electrospinning, nanofibers, phosphopeptide enrichment, phosphoprotein, protein fractionation

*Corresponding author Dr. Wenwan Zhong, Associate Professor Department of Chemistry, University of California, Riverside Email: [email protected] Phone: 951-827-4925 Fax: 951-827-4713

ACS Paragon Plus Environment


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Abstract:

Phosphorylation is one of the most important post-translational modifications in proteins. Their essential roles in regulation of cellular processes and alteration of protein-protein interaction networks have been actively studied. However, phosphorylated proteins are present at low abundance in cells, and ionization of the modified peptides is often suppressed by the more abundant species in mass spectrometry. Effective enrichment techniques are needed to remove the unmodified peptides and concentrate the phosphorylated ones before their identification and quantification. Herein, we prepared the ZrO2 nanofibers by electro-spinning, a straightforward and easy fabrication technique, and applied them to enrich the phosphorylated peptides and proteins. The fibers showed good size homogeneity and porosity; and could specifically bind to the phosphorylated peptides and proteins, allowing their separation from the unmodified analogs when present in either the simple protein digests or the highly complex cell lysates. The enrichment performance was superior to the commercially available nanoparticles. Moreover, modifying the solution pH could lead to selective adsorption of proteins with different pI values, suggesting the fibers’ potential applicability in charge-based protein fractionation. Our results support that the electrospun ZrO2 nanofibers can serve as a versatile tool for protein analysis with high easiness in preparation and handling.


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1. Introduction Post-translational modifications (PTMs) greatly expand the structural and functional diversity of the proteome.

Among all PTMs, phosphorylation is of significant importance

because it participates in diverse cellular processes, including cell development and proliferation, immune response, metabolism, et al.1-2 Thorough examination of protein phosphorylation in cells can help improve our understanding on how the modification is regulated and how it controls cellular activities, which may lead to discovery of new disease therapies.2 Substantial research efforts have been devoted to improve separation and identification of phosphoproteome by chromatographic techniques and mass spectrometry.3-8 Still, this remains a challenging task, because phosphopeptides are present at very low abundance -- typically less than 5% in a complex protein digest, and their MS signals could be suppressed by the non-phosphorylated ones. One effective solution is to selectively enrich the phosphorylated proteins or peptides prior to MS detection.9-10 Antibodies or phosphoprotein binding domains (PBDs) have been employed to isolate phosphorylated proteins or peptides with high specificity.11-12 However, both are not readily available and expensive to obtain. The more universal and affordable approach takes advantage of the strong coordination between metal ions and phosphates, as done in immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography.10,

13

To simplify

sample handling and eliminate the use of chromatographic instruments, titanium (IV) oxide microspheres or strong anion-exchange monolithic capillaries have been prepared to extract the phosphorylated peptides and proteins.14-17 In addition, magnetic particles have been doped with metal oxides or metal chelators so that they can be easily handled with a magnet.18-24 Moreover, affinity groups for phosphorylated peptides has been used to decorate the matrix assisted laser



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deposition and ionization (MALDI) plates for direct coupling of the sampling and MS analysis steps with no need of peptide elution and liquid transfer.25-27 Such platforms are straightforward in usage but could be difficult to fabricate. Zr(IV) could coordinate with phosphate,28-29 and this property can be employed for enrichment of phosphoproteomes. Compared to other metal oxides used for this purpose, zirconia offers many advantages, including excellent thermal and chemical stability, high strength, low thermal conductivity and high corrosion resistance. In addition, its amphoteric property makes it possible to control phosphate binding or release by tuning the solution pH. Typically, binding between phosphate and Zr(IV) takes place at a low pH of 2-3. At this pH, the most common interference, the non-phosphorylated acidic proteins or peptides, would not bind due to protonation of the carboxyl groups, which could also bind to Zr(IV) with lower affinity. As a result, ZrO2 is considered as a highly selective and robust material for enrichment of phosphoproteomes. As-prepared ZrO2 nanoparticles have been used to selectively enrich or separate phosphopeptides, either in the particle form, packed in microtips, or as the coating on the capillary wall30-34. Alternatively, ZrO2-doped magnetic beads and mesoporous composites have been fabricated, showing the advantage of large surface area-to-volume ratios in facilitating target adsorption.35-36 Compared to spherical particles, long fibers possess high aspect ratios and thus provides even larger specific surface areas. They can be easily produced by electrospinning, a highly simple and versatile way to transform polymer solutions into nanofibers with large varieties in morphology, porosity, diameter, and structure.37-39 Other materials, like ceramics, carbon nanotubes, metal nanoparticles, etc., can also be included during electrospinning to diversify fiber compositions.40-41 Nanofibers have been widely employed in biomedical research to


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produce regenerated tissues, act as cell culture scaffold, and deliver drugs with controlled release capability.37, 42 Electrospun nanofibers often have high porosity, which in addition to their large aspect ratio render high surface area for material adsorption. The long fibers, although with diameters in tens or hundreds nanometers, can be easily removed from suspension by filtration or centrifugation. These features make nanofibers ideal materials for biomolecule enrichment. Although various electrospun nanofibers have been applied to isolate metal ions, organic pollutants, peptides, and proteins from environmental and biological samples,43-46 preparation and application of ZrO2 nanofibers for enrichment of biomolecules is not found. Herein, we report the ZrO2 nanofibers prepared by electrospinning and their applications in enriching phosphorylated peptides as well as in protein fractionation. Compared to the existing nanomaterials, the ZrO2 nanofibers reported herein can be prepared and handled with great easiness. Under the optimal loading and elution conditions, they could enrich both the mono- and multi-phosphorylated peptides from complex protein digests with good purity. Moreover, distinct from previous studies that apply the ZrO2-based nanomaterials to assist with only analysis of phosphorylated proteins, our present study revealed the capability of ZrO2 nanofibers in selective adsorption of acidic, neutral, or basic proteins in loading buffers with different pH. This property can be further utilized to develop zirconia-fiber-based extraction devices for protein separation. Our study supports that such nanofibers are a powerful and versatile enabling tool for proteomic study.

2. Results and Discussion Fabrication and characterization of nanofibers. Zirconia nanofibers were fabricated by electro-spinning the zirconia/polymer sol-gel solution.39 Detailed synthesis procedure can be



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found in Supporting Information. Briefly, 1.5 g zirconium acetylacetonate was mixed with 0.15 g PVP in 2.5 mL ethanol and 1 mL acetic acid. This solution was stirred at 60 °C for 30 min, and then sprayed from a syringe with a 22 gauge blunt needle at a flow rate of 1.0 mL/hr. The electro-spinning voltage was 22 kV. The fibers were collected onto an aluminum foil placed 15 cm away from the spraying needle tip, and then calcinated in air at 550 °C for 6 hrs. The morphology of fabricated nanofiber was observed via SEM and TEM. Figure 1A displays the SEM image of the ZrO2 nanofibers. The diameter of the fibers ranged from 80 to 240 nm, with the average of 150 ± 31 nm (Fig. 1B), which was similar to previously reported in literature.47 The fibers were smooth and extremely long (Fig. 1C). We measured the specific surface area of the nanofibers by the Brunauer-Emmett-Teller (BET) test. The BET surface area was found to be 20.7 m2/g, agreeing with what was reported in the literature for ZrO2 or TiO2 nanofibers.28, 48 (Supporting Information, Figure S1A). Fitting the data to the N2 adsorption/desorption isotherm (Fig. S1) revealed that, the fibers in fact had many nano- and/or meso-sized pores throughout the surface: the pore size ranged from a few nm up to 20 nm with the peak value of 10 nm (insert in Fig. S1A). Further examination by TEM (Fig. 1D) also confirmed the presence of the fine pores on fiber surface. The pores within such a size range could result from evaporation of the PVP embedded in the ZrO2 network during calcination, would increase the surface area of the electrospun nanofibers, and are suitable for analysis of biomolecules like peptides and small proteins. In order to see whether the nanofibers would provide a better surface structure compared to other ZrO2 nanomaterials, we purchased the ZrO2 nanopowder (diameter < 100 nm) from Sigma and did the same measurements. The nanopowder had a BET surface area of 11.8 m2/g and larger pore sizes which ranged from 10-50 nm with a peak value around 40 nm. The


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higher specific surface area of the nanofibers compared to nanoparticles with equivalent or even smaller diameters can enable rapid adsorption of the target materials. Specific extraction of phosphopeptides from standard protein mixtures and cell lysates. The electrospun ZrO2 nanofiber was then applied to extract phosphopeptides. Briefly, the peptide sample was re-dissolved in the extraction solution and mixed with ZrO2 nanofiber. The optimal extraction solution was found to be the one with 3% TFA in 50% acetonitrile (optimization data not shown). The strong acidity of the TFA solution could prompt binding of the deprotonated phosphate groups to the zirconium ions via electron donor-acceptor interaction; but prevent non-specific binding from the interfering carboxyl groups, which would be protonate at pH < 4. After removing the unbound peptides, the nanofibers were washed by the extraction buffer for four times to remove the non-specific binding peptides. Finally, the phosphopeptides were eluted and lyophilizied for further analysis. Elution of the phosphopeptides was done in 5% NH3· H2O. Using the protein digest prepared from the 1:10 (molar ratio) mixture of β-casein and BSA, we tested the enrichment effect with the fibers pre-washed by NaOH. The base activation could dissolve the residual debris left on the fiber surface after calcination, and expose more zirconium ions to the surface for phosphate coordination. Indeed, overnight treatment with 1 M NaOH produced a cleaner mass spectrum for the extracted phosphopeptides (Fig. S2, Supporting Information). We also optimized the amount of fibers used to extract a fixed amount of protein mixture. For 1 pmol β-casein digest, at least 5 µg of the ZrO2 fibers were required to specifically isolate the phosphopeptides from the high background of the non-phosphopeptides (Fig. S3, Supporting Information).



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Under the optimal binding and elution conditions mentioned above, we analysed the peptides extracted from various β-casein and BSA mixtures by MALDI-TOF-MS. Four washes were performed before peptide elution to ensure high purity of the recovered peptides. Before enrichment, due to the existence of a large number of non-phosphorylated peptides present in the sample, only one phosphopeptide with low relative intensity was detected (Figure 2A) in the 1:1 (molar ratio) β-casein : BSA peptide mixture. After enrichment with 250 µg nanofibers, four phosphopeptides were identified (Fig. 2B), providing the full coverage of all 5 phosphorylation sites in β-casein (Table S1, Supporting Information); and the signals from the nonphosphorylated peptides were not noticeable. This result supports that, the ZrO2 nanofibers indeed provide high specificity for enriching the phosphopeptides. Similar phosphopeptide peak intensity and cleanness of the spectrum were maintained with 10 fold more BSA in presence while keeping the β-casein at the constant amount of 50 pmol. When the BSA content continued to increase, i.e. in the 1:100 β-casein : BSA mixture, the signal intensity from the larger peptides, in particular, β3, dropped. The high content of the non-phosphorylated peptides could have covered most of the adsorption sites on the fibers, preventing the binding from the lower abundant phosphopeptides. Still, all five phosphopeptides were detectible, compared to the notenriched sample shown in Fig. 2A. Surprisingly, four peaks with the m/z values matching the four phosphopeptides from α-casein (Table S2, Supporting Information) were found in the spectra (Fig. 2B-D), but with very low relative intensity. They should be from the trace level of α-casein contamination presenting in the commercial β-casein protein. Their presence provides a strong evidence for the high selectivity of the nanofibers over phosphopeptides: even those present as impurity could be specifically extracted.


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Moreover, the selectivity of ZrO2 nanofiber towards phosphopeptides was compared with that of the commercial TiO2 microsphere (diameter of 5 µm, from GL Science Inc., Japan) using the α-casein digest. TiO2 is a more widely applied material than ZrO2 in study of protein phosphorylation.26-27, 48 The ZrO2 nanopowder used in surface area comparison was very difficult to handle in biological samples, although they were made with the same material as the nanofibers. Thus we did not continue to test the capability of the ZrO2 nanopowder in phosphopeptide enrichment. Alpha-casein contains more phosphopeptides with multiple phosphorylation sites than β-casein, more suitable for evaluation of how the extraction materials handle various phosphorylation situations. For direct analysis, five phosphopeptides were identified from 10 pmol α-casein, including three mono-phosphorylated peptides and two di-phosphorylated ones (Figure 3). The m/z range for most peptides was from 1,000 to 2,000. The phosphopeptides showed lower relative intensity than their non-phosphorylated counterparts. Among them, the highest relative intensity was 11% and it was generated from the monophosphopeptide, α6. The intensity of other phosphopeptides was no more than 10%. After enrichment by 125 µg ZrO2 nanofibers, the relative abundance of the phosphorylated peptides increased by 2 times for α9 and up to almost 20 times for α5, with α6 still being the dominant peak and two other monophosphopeptides (α2 and α4) having the relative intensity higher than 80%. A higher nanofibers to protein ratio (12.5 µg fiber vs. 1 pmol protein) was used here than in β-casein extraction to accommodate the higher numbers of phosphorylation sites in α-casein. In addition to α5, other multi-phosphorylated peptides were enriched by the ZrO2 fibers. They were the di-phosphorylated α7 and α 8, as well as the penta-phosphorylated α10. The low intensity of α10 may be caused by the poor ionization efficiency due to the existence of five phosphate groups. On contrary, only α2, α4, and α6 were



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detected with significant intensity in the sample enriched by the TiO2 microsphere. This contradicted to the previously reported results that showed TiO2 was more selective than ZrO2 for multiphosphorylated peptides when both were used to prepare the microtips.32 Besides the strong acidity of TFA used in our work largely suppressed the non-specific binding and enabled effective enrichment of all phosphopeptides on the ZrO2 surface, the large surface area provided by the nanofibers compared to the microspheres may also play a role in yielding sufficient collection of the phosphopeptides, including the multiphosphorylated ones. The high selectivity and enrichment factor over phosphopeptides provided by the ZrO2 nanofibers enabled specific detection of phosphorylated peptides from the tryptic digest of a highly complex biological sample, the cell lysate. About 1 mg of fibers were incubated with 20 µg of the cell lysate prepared from the Raw BlueTM Macrophage cells. The extraction and elution conditions were the same as those used in the above study. In parallel, the Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit was employed to extract the phosphopeptides from the same sample with the procedure recommended by the manufacturer and solutions included in the kit. Database searching result shows that without enrichment, only 16 phosphorylation sites on 8 unique phosphoproteins were identified (Table S3, Supporting Information). After ZrO2 nanofiber enrichment, 123 unique phosphopeptides with 133 phosphorylation sites from 75 proteins were identified by LC-MS/MS (Table S5, Supporting Information). Among them, 115 phosphopeptides can be found in the database of PhosphoSite.4950

On contrary, only 72 phosphosites located at 46 peptides from 30 phosphoproteins were found

from the enrichment by the commercial TiO2 microbeads (Table S4, Supporting Information). Higher specificity was also obtained with the ZrO2 nanofibers: about 83% of the peptides


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extracted by the ZrO2 fibers were phosphopeptides, much higher than that recovered by the magnetic TiO2 microbeads, which was about 20%. Extraction of phosphoproteins and pI-based protein fractionation. Moreover, we assessed the utility of ZrO2 for specific enrichment of phosphorylated proteins. Beta-casein which has up to five phosphorylation sites and ovalbumin that could contain up to two phosphorylation sites were mixed with the non-phosphorylated proteins -- bovine serum albumin (BSA), myoglobin (Myt) and cytochrome c (CytC) -- at an equal amount of 2.5 µg. Because proteins are much larger than peptides, and the phosphorylation sites could be hidden and not readily accessible by the nanofibers, before incubation, 8 M urea was employed to unfold the proteins and expose the phosphorylated sites. Then the denatured proteins were incubated with 1 mg ZrO2 nanofibers for 1 hr. The adsorption capacity of ZrO2 nanofiber towards protein was indirectly examined by measuring the reduction of protein amount in the supernatant by SDSPAGE (Figure 4A). Similar to phosphopeptides, under acidic condition (0.1% TFA with a pH of 2), the phosphorylated proteins could be adsorbed to the fiber --- the band intensity for ovalbumin and β-casein dropped significantly in the supernatant after fiber incubation and removal (Fig. 4 Lane S and pH 2). The smear bands shown below BSA but not at the exact positions as ovalbumin and β-casein could be from aggregation of other proteins at such a low pH. This result demonstrates that the ZrO2 could also specifically extract phosphorylated proteins under proper conditions. Protein adsorption in binding buffers with pH higher than 2 was also investigated. We found that the protein would reach the maximum absorptivity at a pH close to its pI value. For example, at pH 4.0, a majority of BSA, which has a pI of 4.6, were bound to and removed by the fiber. Adsorption of myoglobin (pI ~ 7.0) started at pH 6.0; and at pH 8.0 CytC (pI ~ 9.0) was



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completely adsorbed (Fig. 4A). At pH 8.0, multiple faint but new bands other than those seen in the standard mix showed up on the gel, probably due to protein aggregation. To see more clearly the relationship of the solution pH and adsorbed protein amount, the band intensities were calculated by ImageJ, and the relative intensity for each protein before and after fiber incubation was converted to the percent adsorption of the protein and used to calculate the adsorbed protein amount. Then, the adsorption capability expressed in the unit of µg protein/mg fiber was plotted against pH (Fig. 4B). This figure shows that, while at pH 2, specific adsorption of the phosphorylated proteins could occur, the non-phosphorylated proteins would be adsorbed as well at higher pHs, depending on their pIs. Such adsorption behaviours could be due to the coexistence of the electron acceptor, zirconium ion, and the electron donor, the hydroxyl group, on the surface of ZrO2 fibers. Under acidic conditions, electron donors including phosphate and carboxyl groups could be adsorbed via binding to the zirconium cations, which are Lewis acids. The fibers then functioned as an anion exchange media. Under the more basic conditions, the hydroxyl groups may exhibit net negative charges and thus attract the basic proteins via their positively charged residues. At such conditions, the fibers turned into a cation exchange media. The versatility of ZrO2 as ion exchange media has also been noticed in column chromatography.51-52 Our results hint the possibility of fractionating the phosphorylated, acidic, neutral, and basic proteins by the ZrO2 fibers using solutions of different pHs. Certainly, further study of the capability of ZrO2 in protein fractionation should be conducted to prove this speculation.

3. Conclusions


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Our proof-of-principle results presented above support that ZrO2 nanofibers prepared with the simple electrospinning method can specifically enrich phosphopeptides from complex mixtures. Our work also reveals the noteworthy feature of ZrO2 as a convertible ion exchange medium that will be useful in protein separation. While the mechanism of phosphopeptide adsorption is governed by the material itself, the fibers can provide several advantages over the conventional particles made from the same materials. Firstly, the long nanofibers possess large surface areas for rapid adsorption of target molecules. Secondly, the nanofibers can be handled with easiness. Besides centrifugation, the fibers can also be filtrated by filters with large pore sizes since they are long and can be kept on top of the filters, granting the possibility of highthroughput extraction using filter plates. On contrary, to remove the nanoparticles with diameters around 100 nm, high centrifugation forces are needed; or, magnetic property needs to be introduced during particle preparation, making the fabrication more complicated. Moreover, the fibers can be produced with low cost, high yield (in our hand, 3-hour electrospinning produced 150-200 mg fibers, which could carry out more than 100 extractions), and good batch-to-batch reproducibility. Scale-up preparation in laboratories is also possible by using the multi-channel syringe pumps. In summary, the electrospun ZrO2 nanofibers are a convenient yet valuable tool for proteomic study.

Assorted Content: Supporting Information Experimental methods and conditions; Table S1 and S2 list the sequence and name of each phosphopeptide on the casein; Table S3-5 list all phosphopeptides identified in cell lysate



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before and after extraction with the ZrO2 nanofibers and TiO2 microspheres. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements This work was supported by NSF CAREER CHE #1057113 to W. Zhong and the UCR Collaborative Seed Grants. The authors were also grateful for the kind assistance from ChihYuan Chen in Prof. Quan Cheng’s lab in SEM analysis.

References: 1.

Mowen, K. A.; David, M. Unconventional Post-Translational Modifications in

Immunological Signaling. Nat. Immunol. 2014, 15, 512-520. 2.

Ruprecht, B.; Lemeer, S. Proteomic Analysis of Phosphorylation in Cancer. Expert Rev.

Proteomics 2014, 11, 259-267. 3.

Engholm-Keller, K.; Larsen Martin, R. Technologies and Challenges in Large-Scale

Phosphoproteomics. Proteomics 2013, 13, 910-31. 4.

Guo, M.; Huang, B. X. Integration of Phosphoproteomic, Chemical, and Biological

Strategies for the Functional Analysis of Targeted Protein Phosphorylation. Proteomics 2013, 13, 424-437. 5.

Mann, M.; Ong, S.-E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Analysis of

Protein Phosphorylation Using Mass Spectrometry: Deciphering the Phosphoproteome. Trends Biotechnol. 2002, 20, 261-268. 6.

Olive, D. M. Quantitative Methods for the Analysis of Protein Phosphorylation in Drug

Development. Expert Rev. Proteomics 2004, 1, 327-341.


Page 14 of 31

Page 15 of 31

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


7.

Paradela, A.; Albar, J. P. Advances in the Analysis of Protein Phosphorylation. J.

Proteome Res. 2008, 7, 1809-1818. 8.

Zeller, M.; Koenig, S. The Impact of Chromatography and Mass Spectrometry on the

Analysis of Protein Phosphorylation Sites. Anal. Bioanal. Chem. 2004, 378, 898-909. 9.

Salovska, B.; Tichy, A.; Rezacova, M.; Vavrova, J.; Novotna, E. Enrichment Strategies

for Phosphoproteomics: State-of-the-Art. Rev. Anal. Chem. 2012, 31, 29-41. 10.

Leitner, A. Phosphopeptide Enrichment Using Metal Oxide Affinity Chromatography.

TrAC, Trends Anal. Chem. 2010, 29, 177-185. 11.

Lewandrowski, U.; Sickmann, A.; Cesaro, L.; Brunati, A. M.; Toninello, A.; Salvi, M.

Identification of New Tyrosine Phosphorylated Proteins in Rat Brain Mitochondria. FEBS Lett. 2008, 582, 1104-1110. 12.

Stokes, M. P.; Farnsworth, C. L.; Moritz, A.; Silva, J. C.; Jia, X.; Lee, K. A.; Guo, A.;

Polakiewicz, R. D.; Comb, M. J. Ptmscan Direct: Identification and Quantification of Peptides from Critical Signaling Proteins by Immunoaffinity Enrichment Coupled with Lc-Ms/Ms. Mol. Cell. Proteomics 2012, 11, 187-201. 13.

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. 14.

Batalha, I. L.; Lowe, C. R.; Roque, A. C. A. Platforms for Enrichment of Phosphorylated

Proteins and Peptides in Proteomics. Trends Biotechnol. 2012, 30, 100-110. 15.

Dong, M.; Wu, M.; Wang, F.; Qin, H.; Han, G.; Gong, J.; Wu, R. a.; Ye, M.; Liu, Z.; Zou,

H. Coupling Strong Anion-Exchange Monolithic Capillary with Maldi-Tof Ms for Sensitive Detection of Phosphopeptides in Protein Digest. Anal.Chem. 2010, 82, 2907-2915.



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

16.

Zhang, Y.; Ma, W.; Zhang, C.; Wang, C.; Lu, H. Titania Composite Microspheres

Endowed with a Size-Exclusive Effect toward the Highly Specific Revelation of Phosphopeptidome. Acs Appl. Mater. Interfaces 2014, 6, 6290-6299. 17.

Zhou, H.; Ye, M.; Dong, J.; Corradini, E.; Cristobal, A.; Heck, A. J. R.; Zou, H.;

Mohammed, S. Robust Phosphoproteome Enrichment Using Monodisperse Microsphere-Based Immobilized Titanium (Iv) Ion Affinity Chromatography. Nat. Protocols 2013, 8, 461-480. 18.

Li, Y.; Zhang, X.; Deng, C. Functionalized Magnetic Nanoparticles for Sample

Preparation in Proteomics and Peptidomics Analysis. Chem. Soc. Rev. 2013, 42, 8517-8539. 19.

Sun, N.; Deng, C.; Li, Y.; Zhang, X. Size-Exclusive Magnetic Graphene/Mesoporous

Silica Composites with Titanium(Iv)-Immobilized Pore Walls for Selective Enrichment of Endogenous Phosphorylated Peptides. ACS Appl. Mater. Interfaces 2014, 6, 11799-11804. 20.

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. 21.

Li, X.-S.; Xu, L.-D.; Zhu, G.-T.; Yuan, B.-F.; Feng, Y.-Q. Zirconium Arsenate-Modified

Magnetic Nanoparticles: Preparation, Characterization and Application to the Enrichment of Phosphopeptides. Analyst 2012, 137, 959-967. 22.

Ma, W.-F.; Zhang, C.; Zhang, Y.-T.; Yu, M.; Guo, J.; Zhang, Y.; Lu, H.-J.; Wang, C.-C.

Magnetic Msp@Zro2 Microspheres with Yolk-Shell Structure: Designed Synthesis and Application in Highly Selective Enrichment of Phosphopeptides. Langmuir 2014, 30, 6602-6611. 23.

Ma, W.-F.; Zhang, Y.; Li, L.-L.; You, L.-J.; Zhang, P.; Zhang, Y.-T.; Li, J.-M.; Yu, M.;

Guo, J.; Lu, H.-J.; Wang, C.-C. Tailor-Made Magnetic Fe3o4@Mtio(2) Microspheres with a Tunable Mesoporous Anatase Shell for Highly Selective and Effective Enrichment of Phosphopeptides. Acs Nano 2012, 6, 3179-3188.


Page 16 of 31

Page 17 of 31

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


24.

Zhao, L.; Qin, H.; Hu, Z.; Zhang, Y.; Wu, R. a.; Zou, H. A Poly(Ethylene Glycol)-Brush

Decorated Magnetic Polymer for Highly Specific Enrichment of Phosphopeptides. Chemical Science 2012, 3, 2828-2838. 25.

Hoang, T.; Roth, U.; Kowalewski, K.; Belisle, C.; Steinert, K.; Karas, M. Highly Specific

Capture and Direct Maldi Ms Analysis of Phosphopeptides by Zirconium Phosphonate on SelfAssembled Monolayers. Anal. Chem. 2010, 82, 219-228. 26.

Qiao, L.; Roussel, C.; Wan, J.; Yang, P.; Girault, H. H.; Liu, B. Specific on-Plate

Enrichment of Phosphorylated Peptides for Direct Maldi-Tof Ms Analysis. J. Proteome Res. 2007, 6, 4763-4769. 27.

Wang, H.; Duan, J.; Cheng, Q. Photocatalytically Patterned Tio2 Arrays for on-Plate

Selective Enrichment of Phosphopeptides and Direct Maldi Ms Analysis. Anal. Chem. 2011, 83, 1624-1631. 28.

Nakane, K.; Matsuoka, S.; Gao, S.; Yonezawa, S.; Kim, J. H.; Ogata, N. Formation of

Inorganic Nanofibers by Heat-Treatment of Poly(Vinyl Alcohol)-Zirconium Compound Hybrid Nanofibers. J. Mining & Metallurgy Sec. B-Metallurgy 2013, 49, 77-82. 29.

Su, M.-Y.; Wang, J.; Yao, P.-J.; Du, H.-Y. High Performance Humidity Sensor Based on

Electrospun Zr0.9mg0.1o2-Delta Nanofibers. Chinese Phys. Lett. 2012, 29. 30.

Gates, M. B.; Tomer, K. B.; Deterding, L. J. Comparison of Metal and Metal Oxide Media

for Phosphopeptide Enrichment Prior to Mass Spectrometric Analyses. J. Am. Soc. Mass Spectrom. 2010, 21, 1649-1659. 31.

Salovska, B.; Tichy, A.; Fabrik, I.; Rezacova, M.; Vavrova, J. Comparison of Resins for

Metal Oxide Affinity Chromatography with Mass Spectrometry Detection for the Determination of Phosphopeptides. Anal. Lett. 2013, 46, 1505-1524. 32.

Kweon, H. K.; Hkansson, K. Selective Zirconium Dioxide-Based Enrichment of

Phosphorylated Peptides for Mass Spectrometric Analysis. Anal. Chem. 2006, 78, 1743-1749.



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

33.

Lee, C.-H.; Huang, B.-Y.; Chen, Y.-C.; Liu, C.-P.; Liu, C.-Y. Zirconia Nanoparticles-

Coated Column for the Capillary Electrochromatographic Separation of Iron-Binding- and Phosphorylated-Proteins. Analyst 2011, 136, 1481-1487. 34.

Zhou, H.; Tian, R.; Ye, M.; Xu, S.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Zou, H. Highly

Specific Enrichment of Phosphopeptides by Zirconium Dioxide Nanoparticles for Phosphoproteome Analysis. Electrophoresis 2007, 28, 2201-2215. 35.

Wan, H.; Yan, J.; Yu, L.; Sheng, Q.; Zhang, X.; Xue, X.; Li, X.; Liang, X. Zirconia Layer

Coated Mesoporous Silica Microspheres as Hilic Spe Materials for Selective Glycopeptide Enrichment. Analyst 2011, 136, 4422-4430. 36.

Nelson, C. A.; Szczech, J. R.; Xu, Q.; Lawrence, M. J.; Jin, S.; Ge, Y. Mesoporous

Zirconium Oxide Nanomaterials Effectively Enrich Phosphopeptides for Mass SpectrometryBased Phosphoproteomics. Chem. Commun. 2009, 43, 6607-6609. 37.

Xie, J.; Li, X.; Xia, Y. Putting Electrospun Nanofibers to Work for Biomedical Research.

Macromol. Rapid Commun. 2008, 29, 1775-1792. 38.

McCann, J. T.; Li, D.; Xia, Y. Electrospinning of Nanofibers with Core-Sheath, Hollow, or

Porous Structures. J. Mater. Chem. 2005, 15, 735-738. 39.

Li, D.; Xia, Y. Electrospinning of Nanofibers: Reinventing the Wheel? Advanced

Materials 2004, 16, 1153-1170. 40.

Zhang, C.-L.; Yu, S.-H. Nanoparticles Meet Electrospinning: Recent Advances and

Future Prospects. Chem. Soc. Rev. 2014, 43, 4423-4448. 41.

Su, Z.; Ding, J.; Wei, G. Electrospinning: A Facile Technique for Fabricating Polymeric

Nanofibers Doped with Carbon Nanotubes and Metallic Nanoparticles for Sensor Applications. RSC Adv. 2014, 4, 52598-52610. 42.

Hu, X.; Liu, S.; Zhou, G.; Huang, Y.; Xie, Z.; Jing, X. Electrospinning of Polymeric

Nanofibers for Drug Delivery Applications. J. Controlled Release 2014, 185, 12-21.


Page 18 of 31

Page 19 of 31

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


43.

Zhu, G.-T.; Chen, X.; He, X.-M.; Wang, H.; Zhang, Z.; Feng, Y.-Q. Electrospun Highly

Ordered Mesoporous Silica-Carbon Composite Nanofibers for Rapid Extraction and Prefractionation of Endogenous Peptides. Chem. - Eur. J. 2015, 21, 4450-4456. 44.

Deng, X.; Li, C.; Kang, X. Enrichment of Lead Element from Air Using Electrospun

Nanofibrous Membrane Prepared by Co-Electrospinning of Dithizone with Polystyrene. Appl. Mech. Mater. 2015, 723, 507-510, 5 pp. 45.

Bagheri, H.; Asgari, S.; Piri-Moghadam, H. On-Line Micro Solid-Phase Extraction of

Clodinafop Propargyl from Water, Soil and Wheat Samples Using Electrospun Polyamide Nanofibers. Chromatographia 2014, 77, 723-728. 46.

Schneiderman, S.; Zhang, L.; Fong, H.; Menkhaus, T. J. Surface-Functionalized

Electrospun Carbon Nanofiber Mats as an Innovative Type of Protein Adsorption/Purification Medium with High Capacity and High Throughput. J. Chromatogr. A 2011, 1218, 8989-8995. 47.

Formo, E.; Yavuz, M. S.; Lee, E. P.; Lane, L.; Xia, Y. Functionalization of Electrospun

Ceramic Nanofibre Membranes with Noble-Metal Nanostructures for Catalytic Applications. J. Mater. Chem. 2009, 19, 3878-3882. 48.

He, G.; Cai, Y.; Zhao, Y.; Wang, X.; Lai, C.; Xi, M.; Zhu, Z.; Fong, H. Electrospun

Anatase-Phase Tio2 Nanofibers with Different Morphological Structures and Specific Surface Areas. J. Colloid Interf. Sci 2013, 398, 103-111. 49.

Hornbeck, P. V.; Zhang, B.; Murray, B.; Kornhauser, J. M.; Latham, V.; Skrzypek, E.

Phosphositeplus, 2014: Mutations, Ptms and Recalibrations. Nucleic Acids Res. 2015, 43, D512-520. 50.

Phosphosite Homepage. http://www.phosphosite.org (accessed Sept. 29).

51.

Habib Ur, R.; Khan, S. A. The Ion Exchange Properties of Monoclinic Zirconia. Phys.

Chem. (Peshawar, Pak.) 1992, 11, 55-60.



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

52.

Yu, J.; El Rassi, Z. Chromatographic Properties of Zirconia-Based Stationary Phases for

Ion Exchange Chromatography Having Surface Bound Cationic Functions. J. High Resolut. Chromatogr. 1994, 17, 705-12.


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Captions for Tables and Figures:

Figure 1. (A) SEM image and (B) the corresponding diameter distribution histogram of the ZrO2 nanofibers with magnification of 50,000. (C) SEM image with magnification of 100,000. (D) TEM image with magnification of 50,000.

Figure 2. MALDI-TOF mass spectra of the digested β-casein and BSA mixture before (A, at 1:1 molar ratio) and after ZrO2 nanofiber enrichment (B-D, at molar ratios of 1:1, 1:10 and 1:100, respectively). The amount of β-casein was kept at 50 pmol; and 250 µg ZrO2 nanofibers was used for each extraction. “*” represents the doubly charged fragment.

Figure 3. MALDI-TOF MS spectra of 10 pmol α-casein digest before (A) and after enrichment with 125 µg of the ZrO2 nanofibers (B) or TiO2 microspheres (C).

Figure 4. SDS-PAGE image (A) and relative band intensity (B) of the equal mass (2.5 µg each) mixture of five proteins and its flow-through after incubation with 1 mg ZrO2 nanofiber at pH 2, 4, 6 and 8. Lane 1 was loaded with the protein mixture (BSA, Oval, β-casein, Myo, CytC), and lane 2-5 were loaded with the flow through at pH 2 (0.1%TFA), pH 4 (50 mM NaAc-HAc), pH 6 (30 mM MES) and pH 8 (50 mM Tris-HCl), respectively.



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TOC Figure:


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Figure 1.



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Figure 2


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Figure 3.



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Figure 4


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Fig.1


(A)

(C)


(B)

(D)


Fig.2 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


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Fig.3



Fig.4

(A)

Lane

S

pH2

pH4

pH6

pH8

(B)

Oval -casein

Myo CytC

BSA,

Oval,

 -cas,

Myo,

CytC

3.0

BSA Adsorption Capacity (g/mg)

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2.5 2.0 1.5 1.0 0.5 0.0

2

4

6

pH


8

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TOC

Peptide Mixture

Protein Mixture

ZrO2 Nanofiber

Phosphopeptide Enrichment ACS Paragon Plus Environment

Protein Fractionation

ZrO2 Nanofiber as a Versatile Tool for Protein ... - ACS Publications

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