Cleavable ICAT Reagent Sample Preparation Guidelines

Isotope coded affinity tag technology is used for studying protein regulation from a variety of protein sources including complex lysates organism and whole cells or more simplified mixtures such as those generated from immunoprecipitation. An important 1st step in conducting a quantitation experiment using the ICAT reagent is to define the experimental sample. Generally less complex samples require a smaller sample to generate a reasonable dynamic range for quantitation analysis. When starting to design an experiment, please consider the steps that can be taken to reduce the unwanted, highly abundant and potentially interfering proteins in your sample preparation. For example, serum/plasma contains BSA (35 cysteines) at 55mg/ml, Igs at 20 mg/ml; Transferrin (40 cysteines) at 4mg/ml. Removal of these unwanted proteins from the sample will enable detection of lower abundant proteins from a smaller sample size. In addition, this will reduce the amount of the labeling reagent needed. Decreasing the amount of post labeling separation can also increase the speed of analysis and increase the dynamic range. The method used to acquire the protein sample will vary with the source of protein. Tissue culture cell lysates can by prepared by hypotonic lysis. Single cell organisms can be frozen in liquid nitrogen and ground in a mortar and pestle. Tissue sample or larger organisms might require a combination of freezing, homogenation and/ or sonication. Protease inhibitors can be used to limit unwanted degradation of proteins during the preparation and sample preparation should be performed at 4°C (on ice) to reduce non-specific proteolytic activity. When starting with a crude lysate you will need to ask yourself a number of questions including: do you require only the aqueous soluble proteins? Do you need to solubilize as many proteins as possible? Do you need to remove some cellular components (e.g. by ultracentrifugation) before solubilistion? Detergents and urea are useful for increasing solubilization. Once you have completed the solubilisation of your sample, centrifuge the sample and use a small portion (minimum 5 uL) of the supernatant and do a protein determination. There are several steps within the labeling and digestion protocols where reagent concentration can hinder or help the reaction go forward. In general you want to start the labeling reaction at pH 8.5 in a suitable buffer like 50mM TRIS. The total salt level should be at or below 50mM. The detergent concentration will depend on the specific detergent you use (0.1% SDS, 2% Octyl glucopyranoside, Triton X-100, NP-40, Tween 20, CHAPS). Fresh urea at 8M is acceptable for labeling. If your sample contains other components, you should use a protein precipitation method. The resulting protein pellet should be dissolved in a labeling compatible buffer. The precipitation step will also remove active protease inhibitors that could interfere with trypsin digestion. Protein can be precipitated from solution by organic solvents. A simple acetone precipitation can be used as follows. Sample and acetone should be at 4°C. Slowly add acetone to protein sample while on ice, 6-8 volumes of acetone to the sample. Gently agitate while adding the acetone. Place at -20°C overnight. Centrifuge and remove supernatant without disturbing the pellet. Do not dry the pellet completely as it could become difficult to re-suspend. The soluble protein concentration should then be tested by a protein assay. Several commercial available BCA and Bradford assay kits are available from Pierce, Sigma and BioRAD. Review the effects of different components on the assay. Perform a standard curve in triplicate and run multiple dilutions of your sample. It is important to have a good protein determination of the soluble sample, as ultimately the quality of your ICAT results will be dependent on the initial sample prep quality and protein determination. The concentration of your sample should be between 1 -2 mg/mL.