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The Proteomics Cookbook

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The Proteomics Cookbook Back to website The Proteomics Cookbook A user-friendly guide on what the Proteomics Core can do for you by Simone Sidoli & the Proteomics Core Beta version 0.6 (August 2020) Index Chapter Page number Welcome 3 Standard identification and quantification of a protein mixture 4 Identification and quantification of selected gel bands 6 Identification and quantification of proteins in detergents, e.g. immunoprecipitations 8 Time-resolved proteomics 10 Comprehensive quantification of histone post-translational modifications 12 Quantification of synthesis/degradation rate of proteomes using pulsed metabolic labeling 14 Protein – RNA interaction analysis to identify nucleic acid binding domains of proteins 16 Identification of protein complexes associated on the chromatin (ChIP-SICAP) 18 Estimation of the turnover rate of post-translational modifications 20 Phosphoproteomics 22 Page 2 Introduction & User guide Welcome! The Proteomics Cookbook is a collection of protocols and ideas of experiments that can be performed with the Proteomics Core. Each “recipe” describes established and robust protocols that provide quantitative data about the proteome of your system. This includes identification and quantification of proteins, but also other interesting aspect of your proteome such as turnover, localization and more. You will notice that each protocol is flanked by a page displaying data graphs. The goal is to assist brainstorming regarding the type of information that are achievable with your experiment. We hope this will help you select the most appropriate protocol and stimulate your creativity, including planning novel experiments that you did not expect were feasible. You are more than welcome to design other experiments not described here; we love new challenges! The members of the Core are here at Einstein to help you and discuss with you potential limitations. User guide Each “recipe” lists the required reagents and the procedure. The color coding of the page header represents the difficulty of the procedure Green: simple and highly robust Yellow: medium complexity, some optimization might be required Red: feasible experiments, but consider a learning curve The Proteomics Core can perform for you sample preparations for a selected number of protocols. However, we strongly encourage all the users to consider preparing the samples within their lab. We assure that none of these protocols are challenging nor require expensive reagents. We are certain that, in the long run, you will appreciate the commodity of being familiar with these simple steps. They will always work best in your own hands, because nobody knows your samples more than you do. It is also very likely that samples ready for analysis might have shorter waiting times for obvious reasons. For the best appreciation of this document, please print it front and back, so you can have side-by-side the protocol (on the left) with the type of results obtained from this type of experiment (on the right). Message from the author Dear everyone, thank you very much for the very warm welcome I received at Einstein! We will work very hard to repay your trust by making all of you very proud of the proteomics we do at the School. The Core is here to assist you and work with you. Please, feel free to contact us to discuss new projects, collaborations and more. I trust this Edition of the Cookbook will not be the last one, as it will grow with all of your new ideas and challenges. Looking forward to work with all of you! Simone Sidoli Page 3 Standard identification and quantification of a protein mixture (in-solution digestion) Ingredients (reagents) - Cell lysis buffer: 6 M urea + 2 M thiourea in 50 mM ammonium bicarbonate - Protease inhibitor cocktail (e.g. Thermo Halt Protease Inhibitor Cocktail, 100X, catalog # 78445) - 50 mM ammonium bicarbonate, pH 8.0 - Dithiothreitol concentrated stock solution (e.g. 1 M) - Iodoacetamide, powder - Trypsin sequencing grade (e.g. Promega, catalog # V5111) Recipe (protocol) Estimated preparation time: 3 hours + overnight trypsin digestion - Collect cells in an Eppendorf tube, or a Falcon tube in case the pellet is more than 50-100 µl. Make sure they have been washed with PBS (otherwise proteins from the media will interfere with the analysis). Recommended amount: 100,000 to 1,000,000 cells - Mix the lysis buffer with the protease inhibitor cocktail. Add the minimum volume possible of buffer with the cell pellet. You can start from ~5 times the volume of the pellet, e.g. 20 µL of buffer. Pipette up and down to lyse the cells. The solution should become viscous due to the DNA. You can freeze and thaw to assist cell lysis. Do not heat or sonicate, because urea reacts with proteins at high temperatures - Optional: If you have a solution excessively viscous, i.e. does not pipette well, you can add 1U of benzonase to digest DNA into individual nucleic acids. Check after 1 hour if the solution is less viscous - Dilute sample with 5 times the initial lysis buffer volume using the following buffer: 50 mM ammonium bicarbonate and 5 mM dithiothreitol (DTT). The solution should be at pH 8 - Leave the solution for 1 hour at room temperature. DTT is a reducing agent. This step opens all protein disulfide bonds, which will assist digestion - Add iodoacetamide to a final concentration of 20 mM, and incubate 30 min in the dark. Try to prepare concentrated iodoacetamide, so you can put a small volume in the final sample. Prepare iodoacetamide fresh right before the incubation, as it is a photosensitive reagent - Add trypsin at an approximate concentration of 1:50 enzyme:sample (w:w) overnight at room temperature. For rapid sample usage, just add 0.1% trifluoroacetic acid (TFA) the following morning; for long term, dry the samples using a SpeedVac centrifuge and store in -80 freezer Estimated costs All reagents adopted in this protocol are cost effective and likely present in other labs. Trypsin is about $100 per 100 µg, so the costs should be contained unless you start from enormous starting materials The Core charges per time. An entire proteome will be run with a 2-3 hours gradient, implying that in a day you can fit around 8 runs. A simpler protein mixture would be run with 1 hour gradient PCA plot – Sample similarity Network of regulated proteins with their abundance (node color) Mock Ad5 HSV VACV 40 “-” “+” “-” “+” “-” “+” “-” “+” Mock 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 1 2 3 1 2 3 1 2 3 1 2 1 2 3 30 0.76 0.77 0.70 0.59 0.75 0.71 0.79 0.61 0.43 0.47 0.50 0.56 0.49 0.49 0.49 0.43 0.44 0.49 0.56 0.56 0.46 0.49 0.49 0.62 0.85 0.76 0.51 0.48 0.48 0.70 0.75 0.55 0.44 0.57 0.50 0.68 0.60 0.47 0.59 0.55 1 0.76 0.81 0.71 0.56 0.77 0.83 0.73 0.66 0.54 0.58 0.58 0.59 0.57 0.61 0.59 0.54 0.56 0.62 0.58 0.61 0.57 0.56 0.57 0.64 0.77 0.73 0.58 0.61 0.59 0.67 0.80 0.56 0.45 0.60 0.51 0.76 0.63 0.63 0.67 0.67 2 20 0.77 0.81 0.73 0.63 0.69 0.71 0.78 0.69 0.34 0.37 0.39 0.44 0.35 0.42 0.36 0.36 0.36 0.46 0.43 0.44 0.35 0.37 0.37 0.65 0.77 0.65 0.41 0.44 0.39 0.82 0.76 0.65 0.33 0.45 0.37 0.77 0.64 0.43 0.57 0.47 3 VACV 0.70 0.71 0.73 0.59 0.73 0.72 0.74 0.74 0.44 0.53 0.49 0.53 0.48 0.54 0.51 0.47 0.46 0.57 0.56 0.59 0.50 0.48 0.53 0.86 0.67 0.71 0.50 0.51 0.49 0.69 0.76 0.68 0.41 0.53 0.53 0.63 0.64 0.52 0.57 0.58 4 “ - 0.59 0.56 0.63 0.59 0.52 0.56 0.70 0.83 0.11 0.17 0.17 0.23 0.17 0.20 0.18 0.08 0.13 0.23 0.27 0.26 0.14 0.20 0.22 0.53 0.66 0.58 0.20 0.22 0.18 0.68 0.68 0.72 0.16 0.29 0.25 0.74 0.82 0.20 0.33 0.28 5 ” 10 0.75 0.77 0.69 0.73 0.52 0.80 0.74 0.65 0.49 0.53 0.53 0.52 0.54 0.58 0.57 0.50 0.49 0.60 0.58 0.56 0.55 0.56 0.53 0.70 0.73 0.88 0.55 0.56 0.56 0.58 0.78 0.55 0.42 0.59 0.50 0.63 0.62 0.54 0.62 0.60 6 0.71 0.83 0.71 0.72 0.56 0.80 0.73 0.68 0.58 0.60 0.61 0.60 0.63 0.64 0.62 0.57 0.62 0.68 0.65 0.65 0.64 0.64 0.61 0.69 0.71 0.74 0.59 0.67 0.65 0.59 0.86 0.55 0.41 0.65 0.51 0.64 0.64 0.68 0.72 0.75 7 (13.15 (13.15 %) 0.79 0.73 0.78 0.74 0.70 0.74 0.73 0.78 0.35 0.43 0.44 0.48 0.39 0.45 0.43 0.37 0.37 0.45 0.50 0.49 0.40 0.45 0.45 0.73 0.81 0.75 0.46 0.44 0.44 0.74 0.81 0.68 0.39 0.50 0.46 0.74 0.70 0.42 0.55 0.50 8 0 0.61 0.66 0.69 0.74 0.83 0.65 0.68 0.78 0.26 0.35 0.31 0.37 0.29 0.35 0.34 0.27 0.27 0.36 0.40 0.41 0.28 0.32 0.35 0.68 0.73 0.70 0.32 0.33 0.31 0.75 0.76 0.84 0.27 0.39 0.37 0.80 0.83 0.27 0.40 0.37 9 0.43 0.54 0.34 0.44 0.11 0.49 0.58 0.35 0.26 0.92 0.90 0.89 0.92 0.91 0.91 0.91 0.92 0.88 0.89 0.91 0.92 0.92 0.92 0.43 0.36 0.42 0.82 0.85 0.86 0.23 0.43 0.21 0.79 0.85 0.80 0.20 0.18 0.68 0.63 0.74 1 0.47 0.58 0.37 0.53 0.17 0.53 0.60 0.43 0.35 0.92 0.90 0.91 0.92 0.92 0.91 0.92 0.91 0.88 0.90 0.92 0.92 0.91 0.93 0.55 0.43 0.48 0.83 0.84 0.85 0.30 0.49 0.26 0.76 0.84 0.79 0.30 0.27 0.69 0.63 0.76 2 -10 0.50 0.58 0.39 0.49 0.17 0.53 0.61 0.44 0.31 0.90 0.90 0.88 0.90 0.89 0.89 0.89 0.90 0.89 0.88 0.89 0.89 0.89 0.89 0.50 0.46 0.50 0.80 0.84 0.82 0.27 0.49 0.23 0.74 0.81 0.75 0.31 0.27 0.69 0.66 0.75 3 Mock Component 2 Ad5 0.56 0.59 0.44 0.53 0.23 0.52 0.60 0.48 0.37 0.89 0.91 0.88 0.91 0.91 0.90 0.90 0.89 0.88 0.90 0.90 0.90 0.89 0.91 0.53 0.47 0.52 0.87 0.83 0.84 0.34 0.51 0.31 0.73 0.82 0.78 0.30 0.29 0.69 0.64 0.76 4 0.49 0.57 0.35 0.48 0.17 0.54 0.63 0.39 0.29 0.92 0.92 0.90 0.91 0.94 0.94 0.92 0.93 0.89 0.89 0.91 0.92 0.91 0.91 0.48 0.41 0.50 0.83 0.86 0.85 0.25 0.46 0.22 0.76 0.84 0.77 0.23 0.26 0.73 0.66 0.78 5 -20 HSV 0.49 0.61 0.42 0.54 0.20 0.58 0.64 0.45 0.35 0.91 0.92 0.89 0.91 0.94 0.95 0.92 0.93 0.89 0.90 0.91 0.92 0.89 0.90 0.52 0.43 0.54 0.82 0.85 0.84 0.34 0.51 0.28 0.73 0.83 0.77 0.30 0.31 0.75 0.70
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