Newly Fabricated Magnetic Lanthanide Oxides CoreâShell

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Newly Fabricated Magnetic Lanthanide Oxides Core-Shell Nanoparticles in Phosphoproteomics Fahmida Jabeen, Muhammad Najam-ul-Haq, Matthias Rainer, Yüksel Güzel, Christian W. Huck, and Guenther Karl Bonn Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504818s • Publication Date (Web): 10 Apr 2015 Downloaded from http://pubs.acs.org on April 19, 2015

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Analytical Chemistry

Newly Fabricated Magnetic Lanthanide Oxides Core-Shell Nanoparticles in Phosphoproteomics Fahmida Jabeen1,2, Muhammad Najam-ul-Haq*1,2, Matthias Rainer2, Yüksel Güzel2,Christian W. Huck2, Guenther K. Bonn2

1

: Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800, Pakistan

2

: Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 80-82, Innsbruck 6020, Austria

* Corresponding Author Dr. M. Najam-ul-Haq Institute of Chemical Sciences Bahauddin Zakariya University Multan 60800 Pakistan Tel.: +92 306 7552653 Email: [email protected]

Keywords:

Lanthanide

Oxides,

Core-Shell

Nanoparticles,

Selectivity,

Phosphopeptide Enrichment, HeLa Cells, Prostate cancer, MALDI-MS.

1 ACS Paragon Plus Environment

Sensitivity,


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Abstract Metal oxides show high selectivity and sensitivity towards mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic coreshell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, non-fat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no non-specific bindings. The selectivity is achieved up to 1:8500 using β-casein:BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the re-use of core-shell nanoparticles upto four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential towards prostate cancer. Thus the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.


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Introduction Among the low abundant proteins, phosphorylated proteins are targeted as phosphoprotein kinases and proteases play role in stabilized living systems.1,2 Cancer and other diseases are linked to phosphorylated proteins, as abnormal phosphorylation results in malfunctioning of cellular activities.3 Phosphopeptides enrichment methods like immobilized metal ion affinity chromatography (IMAC)4,5,6 and metal oxide affinity chromatography (MOAC)7,8have been introduced. MOAC is in focus because of the selectivity of metal oxides for phosphopeptides. 9 , 10 All these affinity probes are high-valence metal oxides like TiO2 11 , ZrO212, La2O313, HfO214, SnO215,Ta2O516, CeO217, NiO18 and metal oxide composites19,20 with strong Lewis acidity. The incorporation of multiple metal ions affect the phosphate affinity by changing the distributions of binding sites or changing the Lewis acidity of material.21 Therefore, it is promising to search for affinity probes in binary or multiple metal oxides/hydroxides. The core-shell based materials have better separation efficiency and selectivity.22 Magnetic core-shell materials are reported in combination with TiO2 23 , ZrO2 24 , Al2O3 25 , CeO2 26 , SnO2 27 ,Ga2O3 28 , Ta2O5 29 and ZnO. 30 Recent reports have shown that metal oxides with different components or structures may perform diversely in phosphopeptide enrichment. For example, TiO2 and SnO2 synthesized from the same approach show overlap results in the phosphoproteomic analysis of HeLa cells31, ZrO2 is more selective to mono-phosphorylated peptides32, Nb2O5 and Ta2O5 have non-specific adsorptions and require excessive washings to remove the acidic peptides.33,34 The selectivity of metal oxides for phosphate group is in competition with acidic residues having carboxylic group.35 Transition metal oxides interact to the negatively charged groups with dependence on sample loading conditions. On the other hand, rare earth metal oxides are hard Lewis acids and prefer to bind with more negatively charged phosphate groups as



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compared to the acidic peptide residues. For example, rare earth-based nanomaterials have good catalytic activity in the de-phosphorylation process however their applicability is not fully explored.22,36 In current study, magnetic core-shell nanoparticles are fabricated in combination with two lanthanide oxides, i.e. lanthanum oxide and samarium oxide. High number of coordination sites, stable bi-dentate complex and the characteristics of being hard Lewis acids allow the synthesized materials to achieve maximum selectivity and sensitivity. The tryptic milk digest is applied for the sequence coverage. Serum and spiked HeLa cell digests are employed as complex biological samples for the enrichment of phosphopeptides.


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Experimental Chemicals and Materials Supporting Information includes the detail of chemicals and materials used.

Fabrication and Characterisation of Magnetic Lanthanide Oxide

Core-Shell

Nanoparticles Commercial Fe3O4 nanoparticles (30 mg) were dispersed in a mixture of 30 mL ethanol and 6 mL water through ultra-sonication for 10 min. Silane precursor (TEOS, 3.3 mM, 0.45 mL) was added to the finely dispersed nanoparticles. The mixture was subjected to ultrasonication for another 20 min. To provide basic condition, 30 mM aqueous ammonia was added and ultrasonicated for 1 hour. Silica coated magnetic nanoparticles were then magnetically isolated. The product was washed with ultrapure water. For the fabrication of lanthanide oxide shell, the metal oxide precursors, i.e. samarium isopropoxide and lanthanum isopropoxide (0.36 mL) were added to Fe3O4@SiO2 suspension in ethanol. The reaction mixture was stirred continuously for 18 hours at room temperature.The magnetic core-shell nanoparticles were then washed with ethanol and water and dried in oven. Solid state FT-IR (Perkin Elmer) was used to analyze the functionalities and reaction completion. Scanning electron microscopy (SEM) was conducted for morphology and the size determination (Electron Micro Probe, JEOL 8100, Au-sputtered samples).

Sample Preparation Protocols for preparation of HeLa cell extract, digestion of biological fluids, samples for selectivity/sensitivity studies and re-use of core shell nanoparticles are given in supporting information.



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Phosphopeptide Enrichment Protocol One mg of core-shell nanoparticles (NPs), (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) were sonicated in 1 mL activation buffer (80% ACN in 0.1% (v/v) TFA). For each enrichment analysis, 10 µL (1 µg/µL) of this slurry was used. The NPs were conditioned with 50 µL of 0.1% (v/v) TFA and incubated with tryptic protein digests (20 µL, 5 µL acidified with 15 µL of 0.1% TFA) for 30 min at room temperature. The particles were magnetically isolated from the unbound content. To remove non-phosphorylated peptides, NPs were washed with 50 µL of 80% ACN in 0.2% (v/v) TFA followed by DHB buffer (110 mg of DHB in 0.5% ACN and 0.5% TFA) and water. The bound phosphopeptides were eluted by 10 µL of 1.5% ammonium hydroxide solution. The eluted fraction was subjected to MALDIMS analysis. For serum and HeLa cell extract, the amount of affinity material was increased to 3 µg/µL to avoid the saturation of affinity sites on core-shell nanoparticles.

MALDI-MS Analysis MALDI-MS analysis and data interpretation are provided in Supporting Information.


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Results and Discussion Fabrication and Mechanism The mechanism of the synthesis of magnetic lanthanide oxide core-shell nanoparticles is shown in Figure 1 to elaborate the hydrolysis and condensation of silica onto magnetic iron oxide nanoparticles. The coating of silica refrain magnetic core to play any part in the enrichment process. Water and ethanol mixture is used as solvent medium where water performs the hydrolysis and ethanol facilitates the homogenous coating of TEOS (Figure 1, Step A). Water washing converts the un-reacted groups to active hydroxyl sites which are required to synthesize the metal oxide shell (Figure 1, Step B). The addition of metal isopropoxide results in the co-polymerization with an inorganic oxide network wherein lanthanum and samarium oxide coating is formed (Figure 1, Step C). However, metal isopropoxide is added slowly to the homogenously dispersed Fe3O4@SiO2 nanoparticles in ethanol, so that alkoxides cannot hydrolyze in water and thus form insoluble hydroxide species which precipitate out from an aqueous medium. The final step involves the addition of water to completely hydrolyze the attached metal alkoxides (Figure 1, Step D). Un-reacted metal isopropoxide hydrolyzes to the insoluble hydroxides which precipitate out from the aqueous medium. The successive hydroxyl groups on Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 bind phosphopeptides through bi-dentate ligand co-ordination (Figure 1, Step E). Lanthanum oxide and samarium oxide are selected because the lighter lanthanides (La-Gd) show better adsorption to phosphate sites as compared to carboxyl complexation which is linked to heavier lanthanides.37 Peptides with carboxyl sites are involved in the non-specific binding of acidic residues. The excessive washing rectifies the issue for most of metal oxides like TiO2 and ZrO2 however, it also increases the loss of phosphopeptides. In case of lanthanide oxides,



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binding to phosphate is dominated at lower pH38 which is therefore maintained to reduce the non-specific bindings during the sample loading. Magnetic core shell nanoparticles are characterised by FT-IR for their functionalities, SEM micrograph (Figure S1) for surface morphology and EDX analytics for purity of composite (see supporting information).

Phosphopeptide Enrichment from Standards and Optimization Lanthanum binds strongly to phosphate (solubility product Ksp = -26.158) and is used for phosphate reduction in open water and to reduce algae overgrowth or, for medical purposes in the form of lanthanum carbonate, to reduce excess phosphate in human body (Fosrenols). In present study we use lanthanum oxide in the form of magnetic core shell nanoparticles to attain high selectivity for phosphopeptides. Sm3+ has high affinity towards phosphate group in case of DNA samples. It electrostatically interacts with phosphate groups at low pH of ~3 and can bind electrostatically along with covalent bonding at slightly higher pH of ~5. 39 Considering these facts alongwith the role of MOAC in phosphoproteomics, these two metal oxides are selected from the lanthanide series. Commercial phosphoprotein standard, α-casein digest is applied for the optimization of enrichment protocol. The results show that in comparison to magnetic Fe3O4, the two core-shell nanoparticles have enriched higher number of phosphopeptides (Figure S2 a-c). The most common acidic peptides at m/z 1267.67 (YLGYLEQLLR, αS1, 106-125) and 1760.78 (HQGLPQEVLNENLLR, αS1, 23-37) are significantly abundant as well as difficult to remove. Therefore excessive washings are carried out which may cause the loss of multi phosphorylated peptides. In case of Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3, the inherent property to prefer phosphate containing peptides over the carboxylic acid peptides, increases the selectivity. Different elution conditions for β-casein are applied using ammonium hydroxide solution (Figure S3 ad).

There

is

difference

of

one

phosphopeptide


at

m/z

2556.7

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(FQS*EEQQQTEDELQDKIHPF, 47-67) and overall increase in the signal intensity using 1.5% NH4OH solution whereas above this concentration (2% NH4OH), there is an appearance of salt content in the matrix-sample co-crystallization process. Therefore 1.5% NH4OH solution is selected for carrying out further studies.

Enhanced Selectivity with Complex Peptide Background Selectivity study is carried out by spiking the β-casein digest in bovine serum albumin (BSA) digest, which creates complex background having the BSA peptides also in the mass range of interest. β-casein digest is spiked as all the phosphopeptides derived from this source are identified with sequence coverage and phosphorylation sites. β-casein to BSA peptide mixtures are prepared using 1 µL of β-casein digest with dilution up to 7000 fold of BSA digest (1:7000). The prepared mixtures are applied to both core shell NPs following the enrichment protocol described in experimental section. In case of Fe3O4@SiO2-La2O3, the maximum selectivity is achieved for the peptide mixture at ratio of 1:5000 (Figure 2; a-c). Even higher selectivity up to 1:7000 is achieved for Fe3O4@SiO2-Sm2O3 (Figure S4A; a-e S4B, a-c). This is attributed to the three ways binding affinity (electrostatic, covalent and bidentate coordination) offered by Sm3+.42 IMAC-Ti4+ with ATP as chelating agent has been reported for selectivity up to 1:5000.40 The selectivity is dependent on the affinity sites, their availability and hydrophilicity of the sorbent. The lanthanides also offer f-orbitals which expand the co-ordination sites to bind more than nine ligands to one single metal ion.41 The identified peptides in peptide mixtures of varying ratio of β-casein to BSA are listed in Table 1. The selectivity of Fe3O4@SiO2-Sm2O3 is further tested by spiking β-casein digest into BSA protein solution in the ratio from 1:5000 to 1: 8500. In comparison to peptide background, the selectivity is enhanced to the complexity increased up to 8000 folds. The five phosphopeptides listed in Table 1 are enriched and detected in eluted fractions (Figure S5 a9 ACS Paragon Plus Environment


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d). In case of 1:8500, three phosphopeptides from β-casein at m/z 2061, 3054 and 3124 are detected (Figure S5 e). The increase in selectivity is attributed to the less interfering BSA protein background in comparison to the peptides in BSA digest.

Atto-Molar Sensitivity Measurement Sensitivity reported in literature for core-shell nanoparticles in phosphoproteomics is summarized in Table S1. Several dilutions of β-casein digest in the range from 1 pmol to 0.001 fmol (1 amole) are applied to Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3. Both of core-shell nanoparticles show sensitivity down to 1 fmol with no loss of phosphopeptides. Figure S6(a-e) shows the MALDI-MS spectra of different concentrations enriched on Fe3O4@SiO2-Sm2O3. Similar results are achieved for Fe3O4@SiO2-La2O3. Moving to lower concentrations, two phosphopeptides at m/z 2061.7 (FQS*EEQQQTEDELQDK, 33-48) and 3122.4 (RELEELNVPGEIVES*LS*S*S*EESITRI, 26-41) are enriched as mono- and multiphosphorylated peptides respectively. Figure 3 (a-b) shows the enrichment sensitivity of both core-shell nanoparticles for 0.5 fmol β-casein concentration. At lower concentration of 0.001 fmol, only mono-phosphopeptide is detected for Fe3O4@SiO2-La2O3 (Figure 3c) whereas Fe3O4@SiO2-Sm2O3 enriches both phosphopeptides (Figure 3d). Thus nature of metal ion plays role in the enrichment efficiency in the form of oxide or composite.

Batch to Batch Reproducibility The reproducibility is tested by using tryptic digest of α-casein as it has high number of phosphopeptides with different phosphorylations (mono-, di-, tri- and tetra- phosphorylated). Figure S7A (a-b) and S7B (a-b) shows the reproducibility for two batches of Fe3O4@SiO2La2O3 and Fe3O4@SiO2-Sm2O3. The spectra reveal no difference in the enrichment potential of the two core-shell nanoparticles. The standard deviation (SD) is calculated for the phosphopeptides and is listed in Table S2. The highest SD value calculated for Fe3O4@SiO2-


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La2O3 is 0.80 (phosphopeptide, α S1-3P) whereas 0.64 for Fe3O4@SiO2-Sm2O3 (phosphopeptide, α S1-1P and α S1-3P).

Re-Usability of Lanthanide Oxide-Core Shell NPs The lanthanide oxide core-shell nanoparticles are tested for their re-use. Both of the magnetic core-shell nanoparticles can be re-used up to four cycles without losing the enrichment efficiency (Figure S8A; a-e and Figure S8B; a-e). After the enrichment, the material is washed thrice with activation buffer (80% acetonitrile in 0.1% TFA, v/v) to remove any retained content.

Phosphopeptide Enrichment from Complex Samples The enrichment efficiency of Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 is also tested using complex mixtures. For that, non-phosphoproteins like lysozyme, cytochrome c, myoglobin and BSA are digested together with α- and β-casein. This generates huge nonphosphopeptide background apart from the non-phosphopeptides derived from α- and βcasein. A number of phosphopeptides from α- and β-casein are enriched and analyzed as eluted fractions for both Fe3O4@SiO2-La2O3and Fe3O4@SiO2-Sm2O3 (Figure S9 a-b). The identified phosphopeptides, their sequence, number of phosphorylation and amino acid position is given in Table S3. The unique di-phosphopeptide at m/z 4292.1 for αS2 (RNAVPITPTLNREQLS*TS*EENSKKTVDMES*TEVFTKKT) is also identified along with the abundant phosphopeptides commonly reported in literature. As a medium complexity level mixture, β-casein is spiked in tryptic digest of dephosphorylated HeLa cell extract at the ratio of 1:2000 (1 µL/2mL). The MS spectrum of raw digest prior to the enrichment is given with no enriched phosphopeptide (Figure S10 a). After applying the sample to Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3, six phosphopeptides are



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identified including two tetra-phosphorylated peptides at m/z 3122.4 and 3187.9 (Figure S10 b-c). For the high complexity, milk digest is chosen as it contains number of phosphopeptides from α- and β-casein. The target is to enrich the rare phosphopeptide derived from κ-casein at m/z 1634.4 (EDS*PEVIESPPEIN, 168-181) as it is usually not identified because of its low abundance and minor phosphorylation. Figure 4 shows the enrichment of nineteen phosphopeptides from α-casein, thirteen from β-casein and one from κ-casein. The sequence coverage for the identified phosphopeptides of α-(αS1 36.44% and αS2 32.80%), β- (23.60%) and κ-casein (7.36%) is given in Table S4. It is pertinent to mention that no desalting is performed before applying the sample for enrichment or before carrying out the MS analysis.

Phosphopeptide Enrichment from Serum Digest Serum digest contains number of peptides including phosphopeptides. Human serum is digested using mixture of Lys-trypsin which gives complete digestion and increases the complexity as both enzymes cleave at selective sites. The enriched phosphopeptides are identified with three online sources (Figure 5 a-b). Mascot, PhosphoSite plus and Phosidaare are explored to identify the phosphopeptides, track their protein ID, confirm phosphorylation sites and sequence. They are listed in Table S5 with sequence, protein name and protein accession number (SwissProt). The identified phosphopeptides are compared to show the overlap between Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 (Table S4). Fe3O4@SiO2Sm2O3 enriches 74 phosphopeptides while some of low abundant phosphopeptides are not enriched by Fe3O4@SiO2-La2O3. The enriched phosphopeptides are related to phosphorylated proteins that are reported as potential biomarkers for various cancers. Characteristeic phosphopeptides derived from proteins which are linked to prostate cancer are shown separately and labelled in Figure S11, S12 and S13. Armadillo Repeat gene deleted in velo-cardio-facial syndrome (ARVCF) has 12 ACS Paragon Plus Environment

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increased expression in prostate cancer and causes disruption of cell adhesion, which may facilitate cancer progression.42 Over-expression of phosphorylated p120-catenin (p120) also known as δ-Catenin, promotes tumor formation and results in enhanced nuclear distribution of both β-catenin and HIF-1α in hypoxic condition contributing to progression of prostate cancer.43 Overexpression of Bcl-2 allows the prostate cancer cells to survive in an androgendeprived environment, and to confer resistance to anti-androgen therapy. Phosphorylation of Bcl-2 eliminates the potential anti-apoptotic effect of Bcl-2 and can be used for the prognosisof prostate cancer. 44 Increased phospho-eIF4E levels correlate with disease progression in patients with prostate cancer. As eIF4E phosphorylation is related to tumorigenesis, eIF4E levels can be used as marker for prediction of early recurrence. 45 Decreased expression of phosphatidyl ethanol amine binding protein 1 (PEBP1 also known as RKIP) may be a prognostic marker in prostate cancer, with low RKIP levels indicating early PSA failure. 46 Microtubule-associated proteins (MAP) are products of oncogenes, tumor suppressors, and apoptosis regulators which emphasize that alteration of microtubule dynamics may be one of the critical events in tumorigenesis and tumor progression. Rational microtubule-targeting cancer therapeutic approaches are ideal including proteomic profiling of tumor MAPs. 47 Kinases and phosphatases involved in post-translational modifications significantly contribute to alternative splicing regulation and to its integration in the complex regulative network that controls gene expression in eukaryotic cells. 48 , 49 Considering the importance of phosphopeptides in relevance to prostate cancer, these core shell NPs can be used to enrich phosphopeptides that can be traced down for diagnosis of prostate cancer.

Phosphopeptide Enrichment from HeLa Cell Extract Tryptic digest of phosphorylated HeLa cell extract contains number of abundant phosphoproteins. Phosphopeptides derived from phosphoproteins are enriched (Figure S14). The phosphopeptides are identified using online database search engines. Their sequence, 13 ACS Paragon Plus Environment


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protein ID and m/z values are summarized in Table S6. A huge number, i.e. 184 phosphopeptides are enriched from HeLa cell extract out of which 44 are multiphosphorylated peptides. Total of 236 phosphorylation sites are identified in which 146 are serine, 46 are threonine and 44 are tryrosine modification sites. The most abundant phosphorylation is observed at serine site which makes 61.8 % of total modification.


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Conclusion The magnetic core-shell lanthanide oxides, Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 are fabricated to attain the selectivity with 8500 fold non-phosphopeptide background. Sensitivity is attained down to 1 attomole (20 µL of sample per 10 µg of NPs) concentration of β-casein. Electrostatic interaction, high co-ordination number and stable bi-dentate ligand chemistry of Fe3O4@SiO2-Sm2O3 has edge in terms of selectivity over Fe3O4@SiO2-La2O3. In serum and phosphorylated HeLa cell extract digest, the phosphopeptides are identified using the online databases. High reproducibility and re-use to four cycles highlight the economic importance of the core shell NPs. The core shell NPs enrich phosphopeptides belonging to proteins relevant to prostate cancer.



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Acknowledgments This work is supported by the Higher Education Commission (HEC) of Pakistan and the University of Innsbruck, Nachwuchsförderung, Austria. Furthermore, the authors declare that they have no conflict of interest.

Supporting Information Available Additional information includes the chemicals and materials; preparation of HeLa cell extract; digestion of standard caseins, biological fluids and HeLa cell extract; selectivity/sensitivity assessment; re-use of core shell NPs; MALDI-MS analysis; characterization (FT-IR, SEM, EDX); MS spectra and tables. This information is available free of charge via the Internet at http://pubs.acs.org.


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Table 1: Identified phosphopeptides in spiked mixtures for selectivity assessment of magnetic core-shell lanthanide oxides. S/N ratio is given in brackets. Identified Peptide Sequence FQSEEQQQTEDELQDK IEKFQSEEQQQTEDELQDK KIEKFQSEEQQQTEDELQD K KHIEKFQSEEQQQTEDELQ DKIHPIF RELEELNVPGEIVESLSSSE ESITR RELEELNVPGEIVESLSSSE ESITR

Phosphopeptides detected in varying spiked β-casein:BSA ratio 1:500 1:1000 1:1500 1:2000 1:5000 1:6000 1:7000 2061.45 2061.56 2061.89 2061.78 2061.74 2061.30 (12.5) (20.5) (20.6) (11.5) (11.4) 2432.76 2432.09 2432.85 2432.66 2432.79 (10.5) (14.7) (14.4) (7.7) (6.3) 2556.65 2556.42 2556.90 2556.53 2556.51 (9.2) (12.0) (16.7) (8.5) (9.8) 2797.83 2797.56 2796.89 2797.65 2797.01 (6.7) (11.6) (6.6) (15.9) (6.4) 3054.77 3054.42 3054.65 3053.98 3054.62 3054.89 3054.72 (7.1) (18.7) (12.3) (16.6) 3122.78 3122.89 3122.73 3122.96 3122.61 3122. 89 3122.90 (27.4) (27.7) (18.4) (46.7) (16.6)



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Figure Captions Figure 1: Schematic illustration of Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 fabrication. Step A: Silanization of Fe3O4 by silica precursor; Step B: Ammonia treatment of surface alkyl groups; Step C: Shell designingby metal isopropoxide precursor; Step D: Hydroxyl surface functionalization; Step E: Phosphopeptide enrichment through metal bi-dentate ligand coordination chemistry.

Figure 2: Selectivity measurement for Fe3O4@SiO2-La2O3 using β-casein: BSA mixtures in different ratios as (a) 1:1500; (b) 1:2000; (c) 1:5000. (B) Detected phosphopeptides enriched by Fe3O4@SiO2-Sm2O3 using β-casein: BSA mixture as (a) 1:5000; (b) 1:6000; (c) 1: 7000. The phosphopeptides are labelled by symbol "β". The arrow indicates the expected phosphopeptide derived from β-casein.

Figure 3: MALDI-MS spectra of enriched phosphopeptides by Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 showing sensitivity down to atto-mole concentration of β-casein for the dilutions (a) and (b) 0.5 fmol; (c) and (d) for 0.001 fmol (1 attomol). The mass range is shown in two panels with first for m/z 2061.56 (FQS*EEQQQTEDELQDK, 33-48, 1P) and second for m/z 3122.81 (RELEELNVPGEIVES*LS*S*S*EESITRI, 26-41, 4P). The star represents the presence of phosphopeptides whereas the absence is shown by the arrow.

Figure 4: MALDI-MS spectra showing phosphopeptide enrichment from tryptic milk digest for (a) Fe3O4@SiO2-La2O3 (b) Fe3O4@SiO2-Sm2O3. The identified phosphopeptides are labelled as α-1, α-2, α-3...; β-1, β-2,β-3.... derived from both variants of α-casein (αS1 and αS2) and β-casein. One of the phosphopeptide identified from κ-casein is also shown (in-set). The peptide sequence and amino acid position are listed in Table S3 with sequence coverage given in Table S4.


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Figure 5: MALDI-MS spectra of tryptic serum digest for (a) Fe3O4@SiO2-La2O3 (b) Fe3O4@SiO2-Sm2O3 using 1 mg/mL of material with 20 µL of acidified sample in 0.1% TFA. The identified phosphopeptides are listed in Table S4 with peptide sequence, Protein ID and m/z values for both of the core shell nanoparticles.



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TOC Graphic


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Figure 1: Schematic illustration of Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 fabrication. Step A: Silanization of Fe3O4 by silica precursor; Step B: Ammonia treatment of surface alkyl groups; Step C: Shell designingby metal isopropoxide precursor; Step D: Hydroxyl surface functionalization; Step E: Phosphopeptide enrichment through metal bi-dentate ligand coordination chemistry. 182x237mm (96 x 96 DPI)

ACS Paragon Plus Environment


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

Figure 2: Selectivity measurement for Fe3O4@SiO2-La2O3 using β-casein: BSA mixtures in different ratios as (a) 1:1500; (b) 1:2000; (c) 1:5000. (B) Detected phosphopeptides enriched by Fe3O4@SiO2-Sm2O3 using β-casein: BSA mixture as (a) 1:5000; (b) 1:6000; (c) 1: 7000. The phosphopeptides are labelled by symbol "β". The arrow indicates the expected phosphopeptide derived from β-casein. 132x152mm (96 x 96 DPI)


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Figure 3: MALDI-MS spectra of enriched phosphopeptides by Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3 showing sensitivity down to atto-mole concentration of β-casein for the dilutions (a) and (b) 0.5 fmol; (c) and (d) for 0.001 fmol (1 attomol). The mass range is shown in two panels with first for m/z 2061.56 (FQS*EEQQQTEDELQDK, 33-48, 1P) and second for m/z 3122.81 (RELEELNVPGEIVES*LS*S*S*EESITRI, 2641, 4P). The star represents the presence of phosphopeptides whereas the absence is shown by the arrow. 122x150mm (96 x 96 DPI)



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

Figure 4: MALDI-MS spectra showing phosphopeptide enrichment from tryptic milk digest for (a) Fe3O4@SiO2-La2O3 (b) Fe3O4@SiO2-Sm2O3. The identified phosphopeptides are labelled as α-1, α-2, α3...; β-1, β-2,β-3.... derived from both variants of α-casein (αS1 and αS2) and β-casein. One of the phosphopeptide identified from κ-casein is also shown (in-set). The peptide sequence and amino acid position are listed in Table S3 with sequence coverage given in Table S4. 128x149mm (96 x 96 DPI)


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Figure 5: MALDI-MS spectra of tryptic serum digest for (a) Fe3O4@SiO2-La2O3 (b) Fe3O4@SiO2-Sm2O3 using 1 mg/mL of material with 20 µL of acidified sample in 0.1% TFA. The identified phosphopeptides are listed in Table S4 with peptide sequence, Protein ID and m/z values for both of the core shell nanoparticles. 139x152mm (96 x 96 DPI)


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