2010 support Facile Synthesis of Copper(II)Immobilized on Magnetic Mesoporous

Supporting Information Wiley-VCH 201069451 Weinheim, GermanyFacile Synthesis of Copper(II)-Immobilized Magnetic Mesoporous Silica Microspheres for Selective Enrichment of Peptides for Mass Spectrometry Analysis**Shasha Liu, Hemei Chen, Xiaohui Lu, Chunhui Deng,* Xiangmin Zhang, and Pengyuan Yanganie_201003602_sm_miscellaneous_information.pdfSupporting informationPart 1. Synthesis section Synthesis of Fe3O4@mSiO2 microspheres Magnetic Fe3O4 particles with mean diameter of 250 nm were first synthesized using high-temperature hydrothermal reaction according to previous report.[1] By using the prepared magnetite particles as seeds, a mesoporous silica shell was formed on the Fe3O4 particle through a surfactant involved solution sol-gel process. 50 mg of as-prepared Fe3O4 and 500 mg of cetyltrimethyl ammonium bromide (CTAB) were first mixed in deioned H2O by ultrasonication for 30 min. Then resultant homogenous dispersion was diluted with 400 mL of deioned H2O and 50 mL of NaOH aqueous solution (0.01M) in a flask and further ultrasonically treated for 5 min. Afterward, the obtained basic dispersion was heated at 60 oC for 30 minutes under mechanical stirring to form a stable dispersion. Subsequently, 2.5 mL of tetraethylorthosilicate (TEOS)/ethanol (v/v:1/4) solution was injected into the stable dispersion, and the dispersion was further rapidly stirred for about 1 min and was let stand for 12 hours.[2] After the coating process, the product was collected by magnetic separation and redispersed in acetone for refluxing at 80 oC to remove the surfactant through extraction. After acetone extraction 5 times, magnetic mesoporous silica microspheres were collected and washed using deioned water at least 5 times and dried at 50 oC in vacuum. Immobilization of Cu2+ ions on the Fe3O4@mSiO2 microspheres The dried Fe3O4@mSiO2 microspheres (30 mg) were redispersed in 30 mL of anhydrous chloroform and stirred in an iced bath for 30 min. 0.2 mL of hexanedioyl chloride was then added into the suspension to allow the reaction between silanol groups and carbonyl chloride groups. The resulting dispersion was further stirred in iced bath for 4 h, and the microspheres were quickly collected by magnetic separation and washed with anhydrous chloroform, and redispersed in fresh anhydrous chloroform (30 mL). To the resultant dispersion, 200 mg of iminodiacetic acid was added. After stirring at room temperature overnight, the microspheres was harvestedand thoroughly washed with ethanol and vacuum dried at 50 oC. Finally, the obtained microspheres (10 mg) were soaked in 1 mL of copper sulfate solution (0.02, 0.1 or 0.2 M) under gentle stirring overnight. The final Fe3O4@mSiO2-Cu2+ microspheres were repeatedly washed with deioned water and vacuum dried for enrichment of peptides.Reference: [1] H. Deng, X. L. Li, Q. Peng, X. Wang, J. P. Chen, Y. D. Li, Angew. Chem. 2005, 117, 2842; Angew. Chem. Int. Ed. 2005, 44, 2782. [2] K. L. Ding, B. J. Hu, G. M. An, R. T. Tao, H. Y. Zhang, Z. M. Liu, J. Mater. Chem. 2009, 19, 3725.Part 2. Enrichment Experiments Materials Standard peptide (Angiotensin II ： Molecular Weight=1046.2 Sequence isAsp-Arg-Val-Tyr-Ile-His-Pro-Phe Purity>95%) was purchased from GL Biochem (Shanghai) Ltd. and Microcystin-LR (MC-LR, Molecular Weight=995.5, Fig. S3) was purchased from Alexis Corporation (San Diego, USA). Albumin Bovine Serum (BSA, fraction V) was obtained from Bio Basic Inc. (Toronto, Canada). The sample of human serum was offered by Shanghai Zhongshan Hospital from a consenting volunteer(35-year-old woman) and urine was obtained from a middle-aged man who’s ill with liver cancer. α-Cyano-4-hydroxy-cinnamic acid (CHCA) were purchased from Sigma. Sequencing grade–modified trypsin was purchased from Promega (Madison, WI). Acetonitrile (ACN) and trifluoroacetic acid were purchased from Merck (Darmstadt, Germany). All of other chemicals were of analytical grade and used as received. All aqueous solutions were prepared using Milli-Q water by Milli-Q system (Millipore, Bedford, MA).Preparation of protein digestion Albumin Bovine Serum (BSA), 1 mg mL-1 in 25 mM ammonium bicarbonate buffer at pH 8.0, were digested with 1:40 Trypsin:Protein (w/w) ratio for 15 h at 37oC.MALDI-TOF-MS process Peptides or proteins were deposited on the MALDI target using dried droplet method. 1 µL of the eluate was deposited on the plate and dried, then another 0.5 μL of a mixture of 4 mg mL-1 CHCA (in 50% (v/v) acetonitrile and 0.1% (v/v) TFA aqueous solution) was introduced. MALDI-TOF MS experiments were performed in positive ion mode on a 4700 Proteomics Analyzer (Applied Biosystems, USA) with the Nd-YAG laser at 355 nm, a repetition rate of 200Hz and an acceleration voltage of 20 kV.Search parameters: Database: SwissProt; digest used: Trypsin; maximum of missed cleavages: 1; mass tolerance: ± 20 ppm. Mascot from Matrix Science Ltd. (London, U.K.) was used to search all of the mass spectra. Protocol of enrichment process First, Fe3O4@mSiO2-Cu2+ microspheres (5 μL, 10 mg mL-1, in water) were added into the solution of standard peptides (400 μL, 5 nM) separately. Then the solution containing peptides and microspheres was shaken at room temperature for 2 min. After disarding the supernatant from suspension with the help of an external magnet, the deposition was rinsed with water for three times; and the peptides retained on the surface of spheres were eluted with the ammonia solution (2 μL, 0.4 M). Finally, the eluate containing the captured peptides was separated from microspheres with the help of magnet. Following 1 μL of the eluate was deposited on the plate and dried, 0.5 μL CHCA was introduced for MALDI-TOF MS Analysis. Similarly, for standard protein digestion, a certain quantity of Fe3O4@mSiO2-Cu2+ microspheres were added into BSA digest solution at the concentration of 5 nM andthe suspension was incubated for 2 minutes. Then the eluted supernatants of microspheres containing enriched peptides was directly analyzed by MALDI-TOF MS. To compare with the result of the eluate, the mass spectra of BSA digest solution of 5 nM without enrichment is shown in Fig. S5. Only 7 peptides in low intensity were detected, While 18 peptides at high intensity were revealed in the spectra (Fig. S5 and Table S1) at a broad range of GRAVY. The results above indicate that Fe3O4@mSiO2-Cu2+ microspheres are effective materials for peptides enrichment at a low concentration.abc80 60 40 20 0 O 61.43 Si 24.7 Fe 9.92 Cu 3.95dMean Value: Atomic%Figure S1. (a) TEM image, (b) SEM image and （ c ） EDX spectrum of theFe3O4@mSiO2-Cu2+ microspheres obtained by soaking the HC-IA modified Fe3O4@mSiO2 microspheres in 0.2 M CuSO4 solution; (d) pattern of mean value of atomic (%) in the microspheres.abFigure S2. Fourier transform Infrared (FTIR) spectrum of (a) Fe3O4@mSiO2 microspheres and (b) Fe3O4@mSiO2 –Cu2+microspheres.Intensity (a. u.)311220 400 511 422 440203040506070802 Theta (degree)Figure S3. Wide-angle X-ray diffraction patterns of the synthesized Fe3O4@mSiO2 -Cu2+ microspheres.6040Magnetization (emu g )-1200-20-40-60 -15000-10000-5000050001000015000Magnetic Field (Oe)Figure S4. Hysteresis loops of Fe3O4@mSiO2-Cu2+ microspheres recorded at room temperature.D-GluH COOH CH 3 NMdhaCH 2ADDAH OCH 3 H HHN H3C H O O O NH H 3C NH H H CH 3 H N HD-AlaOHCH 3CH 3H[R 2 ]H[R 1 ]D-β -Me-AspOCOOHFigure S5. The configuration of cyclopeptide Microcystin-LR (MC-LR, R1 denotes leucine and R2 denotes arginine in the structure)Figure S6. MALDI-TOF mass spectra of 5 nM Angiotensin II ((Mw=1046.2, isoelectric point (pI)=6.74)) (a) before enrichment and after enrichment by (b) Fe 3O 4@m SiO 2 microspheres, (c) organic spacer modified Fe 3O 4@m SiO 2, (d) Fe 3O 4@m SiO 2-Cu 2+ microspheres by soaking in 0.02 M CuSO4 solution, (e) Fe 3O 4@m SiO 2-Cu 2+ microspheres by soaking in 0.1 M CuSO 4 solution, (f) summary of the enrichment factor attained by using different absorbents.abcE n r i c h m e n t f a c t o rAbsorbentdeabFigure S7. a) EDX spectrum of the Fe3O4@m SiO2-Cu2+ microspheres obtained by soaking the HC-IA modified Fe3O4@m SiO2 microspheres in 0.1 M CuSO4 solution and b) pattern of mean value of atomic (%) in the microspheres.abFigure S8. MALDI-TOF mass spectra of 5 nM BSA digest solution a) without any treatment; b) enriched by Fe3O4@m SiO2-Cu2+ microspheres. (The mark “*” in mass spectra image denotes peptide which can be find in database.)abFigure S9. MALDI-TOF mass spectra of diluted human urine solution a) without any treatment; b) enriched by Fe3O4@m SiO2-Cu2+ microspheres.Table S1. The results of a 5 nM BSA tryptic digestion after enrichment byFe3O4@m SiO2-Cu2+ microspheres.Observed m/z Database sequence GRAVY-0.950 712.4125 SEIAHR-1.014 847.5402 LSQKFPK-0.071 927.5400 YLYEIAR-1.425 974.5063 DLGEEHFK0.0201050.4874 EACFAVEGPK0.1901142.7690 KQTALVELLK0.1301163.6906 LVNELTEFAK-1.700 1193.6616 DTHKSEIAHR-1.250 1249.6876 FKDLGEEHFK0.2641283.7800 HPEYAVSVLLR-0.582 1305.7850 HLVDEPQNLIK-0.133 1439.8839 RHPEYAVSVLLR0.2921479.8807 LGEYGFQNALIVR0.2071511.9240 VPQVSTPTLVEVSR-0.085 1567.8219 DAFLGSFLYEYSR-1.723 1616.8407 QEPERNECFLSHK-0.067 1640.0350 KVPQVSTPTLVEVSR1823.9972 RPCFSALTPDETYVPK-0.537 Peptides matched 18Sequence coverage (%) 25%。