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[…] Exponentially growing cells were collected, washed in cold water and lysed using glass beads in 10 mM Tris-HCl pH 7.4, 140 mM NaCl, 1.5 mM MgCl2 and 0.2% NP40. 40 mg of total protein were used for IP with 40 μg Flag antibody and Protein A Dynabeads for 6 hr at 4°C. After 4 washes in lysis buffer, proteins were eluted in 200 μl of 300 μg/ml 3X Flag peptide for 30 min, at room temperature. Eluted samples were TCA-precipitated, resuspended in 8 M urea, and treated with ProteasMAX (Promega, Madison, WI) per the manufacturer’s instruction. Subsequently, samples were reduced by 20 min incubation with 5 mM TCEP (tris(2 carboxyethyl)phosphine) at room temperature, alkylated in the dark by treatment with 10mM Iodoacetamide for 20 min, and quenched with excess TCEP. Proteins were digested overnight at 37 degrees with Sequencing Grade Modified Trypsin (Promega) and the reaction was stopped with formic acid.The protein digest was subjected to cation-exchange microcapillary chromatography, and elutates from the column were electrosprayed directly into an LTQ mass spectrometer (ThermoFinnigan, Palo Alto, CA). A cycle of one full-scan mass spectrum (400–2000 m/z) followed by 7 data-dependent MS/MS spectra at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. Application of mass spectrometer scan functions and HPLC solvent gradients were controlled by the Xcalibur datasystem.Protein identification and quantification analysis were done with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications, Inc. San Diego, CA). Tandem mass spectra were searched against the Saccharomyces Genome Database (SGD) protein database (http://www.yeastgenome.org/download-data/sequence, released on 01-05-2010). LTQ data was searched with 3000.0 milli-amu precursor tolerance and the fragment ions were restricted to a 600.0 ppm tolerance. The ProLuCID search results were assembled and filtered using the DTASelect program (version 2.0) (; ) with false discovery rate (FDR) of 0.15, under such filtering conditions, the estimated false discovery rate was less than 5.4% at the protein level in each analysis. […]

Pipeline specifications

Software tools ProLuCID, DTASelect
Application MS-based untargeted proteomics
Diseases Immunoproliferative Small Intestinal Disease