Supporting Information. for. Advanced Materials, adma Wiley-VCH 2006
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1 Supporting Information for Advanced Materials, adma Wiley-VCH Weinheim, Germany
2 Supporting Information Synthesis of Magnetic Microspheres with Immobilized Metal Ions for Enrichment and Direct Determination of Phosphopeptides by MALDI-TOF- MS/MS Xiuqing Xu, Chunhui Deng*, Mingxia Gao, Wenjia Yu, Pengyuan Yang, Xiangmin Zhang* Department of Chemistry & Center for Proteomics Research, Fudan University, Shanghai , China *Corresponding author, Dr. C. Deng, and Prof. X. Zhang and Fax:
3 Experimental Section Part A: preparation and characterization of magnetic microspheres Synthesis of magnetic microspheres The magnetic microspheres were synthesized through solvothermal reaction. described as following: 2.70 g of FeCl 3 6H 2 O were first dissolved in 100 ml of ethylene glycol under magnetic stirring. A yellow clear solution was obtained after stirring for 0.5 h. Then 7.20 g of NaAc (sodium acetate) were added to this solution. After stirring for another 0.5h, the resultant solution was transferred into a Teflonlined stainless-steel autoclave with capacity of 200 ml. The autoclave was sealed and heated at 200 o C for 8 h, and cooled to room temperature. The black magnetic microspheres were collected with the help of magnet, followed by washing with recycle of ethanol and deioned water for 6 times, then the product was dried in vacuum at 60 o C for 12 h. Synthesis of magnetic silica microspheres In order to obtain core-shell MS microspheres with narrow size distribution and uniform thickness of silica via sol-gel approach, magnetic microspheres (0.01 g) were first treated in HCl aqueous solution (5.0 ml, 2 M) under ultrasonic vibration for 5 min. Then the microspheres were thoroughly washed with deioned water and redispersed in a mixture of ethanol (70.0 g), deioned water (20.0 g) and concentrated ammonia aqueous solution (1.0 g, 28 Wt.-%) with the help of ultrasonication, and a stable dispersion was obtained. Subsequently, tetraethyl orthosilicate (0.05 g) was added to the above dispersion under mechanistically stirring and the reaction was allowed to proceed for 12 h. Finally, by use of a magnet, the product was separated and washed with ethanol and water, and then vacuum dried at 60 o C for 24 h. Immobilization of Fe 3+ onto MS microspheres (i) Synthesis of GLYMO-IDA silane coupling agent
4 In a 100mL flask bottle, iminodiacetic acid (IDA, 4.20 g) was dissolved in sodium hydroxide aqueous solution (10 M), and the solution (50 ml) was adjusted to ph 11. After placing the reactor in an ice-bath at 0 o C and magnetic stirring for 1.0 h, 3- Glycidoxypropyltrimethoxysilane (GLYMO, 1.5 g) was added dropwise under magnetic stirring within 0.5 h. Then, the mixture was heated to 65 o C for 6 h reaction, and then the reaction system was cooled to 0 o C in ice-bath. In order to ensure the reaction between IDA and GLYMO and avoid the crosslinking reaction between GLYMO molecules, the above procedure was repeated twice including addition of GLYMOreaction at 65 o C for 6h and cool-down in ice-bath at 0 o C. Finally, the obtained IDA-derived silane coupling agent solution was adjusted to ph 6 with concentrated HCl for next grafting reaction on the surface of MS micropsheres. (ii) Immobilization of Fe 3+ onto MS microspheres 0.02 g of MS microspheres powder were first dispersed in 50.0 g of ethanol with the aid of ultrasonication, then, 10.0 g of silane coupling agent solution was added under continuously mechanical stirring, and the reaction was allowed to proceed for 24 h at 40 o C. Finally, the product was collected and thoroughly washed with ethanol. To further immobilize Fe 3+ on these obtained microspheres, the product was redispersed in an aqueous solution of FeCl 3 (20 ml, 0.2 M), and the dispersion was stirred for 2 h. Thereafter, the excessive Fe 3+ was removed by repeated washing with deioned water and vacuum dried overnight at 60 o C, and Fe 3+ immobilized MS microspheres were prepared. Characterization: Transmission electron microscope (TEM) images and Scanning electron microscope (SEM) images were obtained on JEM-2100F (JEOL) and XL30 (Philips) respectively. The FT-IR spectrum of MS microspheres and Fe 3+ -MS microspheres were recorded on FT-IR (NEXUS470, NICOLET).
5 Part B: Protein Enrichment experiment Materials Bovine β-casein, Phosphoric acid (H 3 PO 4 ), Ammonium bicarbonate (NH 4 HCO 3 ) and 2, 5-dihydroxybenzoic acid (2, 5-DHB) were purchased from Sigma Chemical (St. Louis, MO). Sequencing grade modified trypsin was purchased from Promega (Madison, WI). Acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). All aqueous solutions were prepared using Milli-Q water by Milli-Q system (Millipore, Bedford, MA). Sample preparation Bovine β-casein, 1mg ml -1 in 25mM ammonium bicarbonate buffer at ph 8.0, was digested with 1:50 Trypsin:Protein (w/w) ratio for 12 h at 37 o C respectively. Enrichment of phosphopeptides by Fe 3+ -MS microspheres Suspension of Fe 3+ -MS microspheres (10 µl of 1 mg ml -1 ) was added into 200 µl of 0.2 nmol ml -1 or 0.02 nmol ml -1 Bovine β-casein peptides solutions respectively. Then the mixed solutions were vibrated at 37 o C for 90 min. After that, with the help of magnet, the peptides/fe 3+ -MS microspheres were collected by removal of the supernate and washed with 20% acetonitrile aqueous solution (v/v) for one time. Finally, the obtained peptides/fe 3+ -MS microspheres were redispersed in 10 µl of 50% acetonitrile aqueous solution (v/v). MALDI-TOF-MS process Peptides/Fe 3+ -MS microspheres were deposited on the MALDI target using dried droplet method. 0.4 µl of the above slurry was deposited on the plate and then another 0.4 µl of a mixture of 30 mg ml -1 2,5-DHB (in 50% acetonitrile aqueous solution, v/v) and 1% (v/v) H 3 PO 4 aqueous solution, 1:1 (v/v) 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.
6 Figure SI Enlarged TEM image of magnetic microspheres, indicating that magnetic microspheres consists of magnetite nanoparticles
7 Figure SII Consecutive photographs of the separation process of magnetic silica microspheres in water (0.1 wt.-%) under applied magnetic field (2000Gs), indicating fast response of magnetic silica microspheres in reply to magnetic field.
8 (b) (a) Figure SIII FT-IR spectra of (a) MS microspheres and (b) Fe 3+ -MS microspheres, from which absorption peaks at ~2900 cm -1 of CH 2 in silane coupling agent and absorption bands at ~1610 cm -1 and ~1400 cm -1 assigned to the carboxylate were observed, confirming the metal ions were successfully immobilized on the magnetic silica microspheres.
9 Figure SIV SEM images of (a) MS microspheres and (b) Fe 3+ -MS microspheres which reveal that the surface modification and Fe 3+ -immolization have no effect on the morphology and dispersibility of magnetic silica microspheres.
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