By Salvatore, Ed. Sechi
Read or Download Quantitative Proteomics by Mass Spectrometry (Methods in Molecular Biology Vol 359) PDF
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Extra resources for Quantitative Proteomics by Mass Spectrometry (Methods in Molecular Biology Vol 359)
B. 32 mg/mL 100X benzamidine in water. Store at –20°C. c. 15 mg/mL leupeptin (500X) in ethanol. Store at –70°C for less than 6 mo. d. 28 mg/mL pepstatin (200X) in methanol. Store at –20°C. e. 5 mg/mL chymostatin (2500X) in dimethyl sulfoxide. Store at –20°C. 2. Immunopurification of Protein Complexes 1. 05% NP-40. Add protease inhibitors before use. 2. 0025% NP-40. 3. FLAG-M2 agarose (Sigma-Aldrich, St. Louis, MO). 4. 3X FLAG peptide (Sigma-Aldrich). 3. Stable Isotope-Labeling With Cleavable ICAT Reagents 1.
3. Peptides are slowly loaded onto equilibrated SCX cartridges (~1 drop/s). Save the flow through. 75 mL SCX buffer containing 40, 200, 350, and 600 mM KCl into Eppendorf tubes (see Note 8). 4. To clean the SCX cartridge, wash with 2 mL SCX buffer containing 1 M KCl. To store the column, wash with 2 mL SCX buffer. 1% sodium azide in the wash. Cartridges can be reused approx 20 times depending on the complexity of the samples. 6. Purifying ICAT-Labeled Peptides 1. 2). 0 with approx 10 µL 1 M ammonium bicarbonate.
Prepare fresh. 3. ) 1. Nanosep 3K centrifugal devices (Pall Corporation, New York, NY). 4. Preparation of Control Peptides 1. 1-cc MCX cartridges (Waters). 3. Methods We describe methods for characterizing the composition of protein complexes and for detecting changes in the composition of complexes using stable isotope tagging and MS. In both approaches, single-step affinity purifications can be used for complex isolation. Owing to the high resolving power of liquid chromatography–mass spectrometry (LC–MS) techniques, the copurifying contaminants are not detrimental.