High throughput functional proteomics
a high-throughput, proteomics technology, applied in the field of integrated systems based on functional affinity chromatography and large-scale protein identification, can solve the problems of time-consuming process and identification of correct functions
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example 1
Preparation of Soluble Rice Protein Extracts
[0070] Thirty to fifty grams of leaves or roots from Oryza sativa (6 flats of 6-week old plants) were snap frozen in liquid nitrogen, and the tissue ground into ice-cold extraction buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 1% sodium deoxycholate, protease inhibitors). Tissue was allowed to solubilize, for 5 minutes with stirring on ice. Crude tissue extract was filtered through cheesecloth (2×), and then miracloth (1×) to remove particulate matter. Insoluble material was spun out at 10,000 g, for 15 minutes at 4° C. The supernatant was then filtered sterilized through a 0.2 micron Nalgene filter, concentrated to at least 10 mg / ml on an Amicon stirred cell using a YM3 molecular weight cut-off membrane, and dialyzed overnight into column equilibration buffer. Aliquots were stored at −80° C.
[0071] In Example 2 functional affinity chromatography is performed.
example 2
Isolation of Carbohydrate Binding Proteins by Functional Affinity Chromatography
[0072] Columns consisting of either D-mannose, N-Acetyl-D-glucosamine (GlcNAc), N-acetyl-galactosamine (GalNAc) or α-L-fucose carbohydrate residues bound to agarose beads were equilibrated in the appropriate buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 for mannose, fucose, and GlcNAc columns, or 20 mM Bis-Tris, pH 7.0, 50 mM NaCl, 0.1% reduced Triton-X 100, 2 mM CaCl2, 2 mM MgCl2, for the GalNAc column) over 5 column volumes. The columns were purchased from E-Y laboratories (San Mateo, Calif.) D-mannose gel, catalog #CG-005-5, N-acetyl-galactosamine gel, catalog #CG-002-5, N-acetyl-glucosamine gel, catalog #CG-003-5, and alpha-L-fucose gel, catalog # CG-001-5. The protein extract of interest was loaded onto the column at a rate of 0.2 ml / minute, and allowed to bind for 30 minutes at 4° C. Bound proteins were then eluted over a continuous gradient of 10 column volumes from 0-100% buffe...
example 3
Preparation of Samples and Identification and Analysis of Proteins Using Mass Spectrometry Analysis
[0074] Peak fractions were analyzed by SDS-PAGE under reducing conditions. FIG. 2A shows proteins from the whole protein extract (lane 1) and the entire protein fraction that binds to the mannose-agarose affinity column (lane 2). FIG. 2B shows proteins present in peak protein fractions isolated from soluble leaf extract after chromatography over a mannose-agarose affinity column (lanes 1 to 5 correspond to fractions 6 to 10 in FIG. 1). Silver-stained protein bands were excised, and the proteins extracted and trypsin digested by incubation overnight at 37° C., then buffer exchanged into 0.1% formic acid preparatory to mass spectrometric analysis by liquid chromatography and tandem MS (LC-MS / MS) using a Finnigan LCQ ion trap mass spectrometer as follows:
[0075] For one-dimensional electrophoresis, gels were run according to established methods using a BioRad mini-gel system and BioRad p...
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