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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 0.79 6 0.49 0.59 0.36 0.51 0.18 0.57 0.62 0.43 0.34 0.91 0.91 0.89 0.90 0.94 0.95 0.92 0.92 0.87 0.89 0.90 0.91 0.90 0.90 0.48 0.42 0.52 0.81 0.83 0.83 0.30 0.47 0.25 0.77 0.83 0.76 0.30 0.32 0.69 0.64 0.74

7 “+” 0.43 0.54 0.36 0.47 0.08 0.50 0.57 0.37 0.27 0.91 0.92 0.89 0.90 0.92 0.92 0.92 0.93 0.85 0.87 0.90 0.90 0.90 0.91 0.47 0.39 0.47 0.82 0.81 0.83 0.26 0.45 0.20 0.79 0.82 0.77 0.26 0.19 0.65 0.60 0.69 8 -30 0.44 0.56 0.36 0.46 0.13 0.49 0.62 0.37 0.27 0.92 0.91 0.90 0.89 0.93 0.93 0.92 0.93 0.89 0.88 0.89 0.91 0.91 0.89 0.45 0.36 0.44 0.80 0.84 0.85 0.27 0.45 0.20 0.77 0.82 0.74 0.23 0.22 0.74 0.69 0.77 9 0.49 0.62 0.46 0.57 0.23 0.60 0.68 0.45 0.36 0.88 0.88 0.89 0.88 0.89 0.89 0.87 0.85 0.89 0.88 0.90 0.90 0.87 0.88 0.56 0.43 0.53 0.79 0.91 0.85 0.34 0.53 0.28 0.72 0.83 0.77 0.30 0.33 0.76 0.74 0.82 10 0.56 0.58 0.43 0.56 0.27 0.58 0.65 0.50 0.40 0.89 0.90 0.88 0.90 0.89 0.90 0.89 0.87 0.88 0.88 0.94 0.90 0.90 0.91 0.54 0.49 0.54 0.81 0.85 0.84 0.32 0.56 0.33 0.71 0.84 0.78 0.32 0.35 0.72 0.69 0.80 11 -40 0.56 0.61 0.44 0.59 0.26 0.56 0.65 0.49 0.41 0.91 0.92 0.89 0.90 0.91 0.91 0.90 0.90 0.89 0.90 0.94 0.92 0.92 0.93 0.54 0.49 0.53 0.82 0.86 0.85 0.34 0.55 0.34 0.75 0.87 0.81 0.33 0.37 0.72 0.66 0.80 12 -30 -20 -10 0 10 20 30 40 50 60 70 0.46 0.57 0.35 0.50 0.14 0.55 0.64 0.40 0.28 0.92 0.92 0.89 0.90 0.92 0.92 0.91 0.90 0.91 0.90 0.90 0.92 0.93 0.93 0.48 0.38 0.48 0.83 0.86 0.89 0.27 0.47 0.24 0.77 0.87 0.80 0.25 0.23 0.71 0.66 0.78 13 Component 1 (19.88 %) 0.49 0.56 0.37 0.48 0.20 0.56 0.64 0.45 0.32 0.92 0.91 0.89 0.89 0.91 0.89 0.90 0.90 0.91 0.87 0.90 0.92 0.93 0.94 0.48 0.42 0.50 0.83 0.85 0.88 0.28 0.50 0.26 0.79 0.90 0.82 0.23 0.28 0.69 0.65 0.76 14 0.49 0.57 0.37 0.53 0.22 0.53 0.61 0.45 0.35 0.92 0.93 0.89 0.91 0.91 0.90 0.90 0.91 0.89 0.88 0.91 0.93 0.93 0.94 0.52 0.45 0.50 0.84 0.85 0.87 0.27 0.51 0.28 0.78 0.88 0.85 0.30 0.32 0.69 0.64 0.76 15

0.62 0.64 0.65 0.86 0.53 0.70 0.69 0.73 0.68 0.43 0.55 0.50 0.53 0.48 0.52 0.48 0.47 0.45 0.56 0.54 0.54 0.48 0.48 0.52 0.63 0.71 0.53 0.51 0.50 0.71 0.70 0.66 0.46 0.52 0.54 0.62 0.63 0.51 0.57 0.58 1

“ -

0.85 0.77 0.77 0.67 0.66 0.73 0.71 0.81 0.73 0.36 0.43 0.46 0.47 0.41 0.43 0.42 0.39 0.36 0.43 0.49 0.49 0.38 0.42 0.45 0.63 0.78 0.49 0.44 0.43 0.69 0.81 0.63 0.37 0.50 0.44 0.77 0.74 0.42 0.54 0.48 ” 2 Ad5 0.76 0.73 0.65 0.71 0.58 0.88 0.74 0.75 0.70 0.42 0.48 0.50 0.52 0.50 0.54 0.52 0.47 0.44 0.53 0.54 0.53 0.48 0.50 0.50 0.71 0.78 0.53 0.49 0.49 0.58 0.77 0.57 0.38 0.54 0.48 0.66 0.67 0.49 0.60 0.54 3 Not identified 0.51 0.58 0.41 0.50 0.20 0.55 0.59 0.46 0.32 0.82 0.83 0.80 0.87 0.83 0.82 0.81 0.82 0.80 0.79 0.81 0.82 0.83 0.83 0.84 0.53 0.49 0.53 0.86 0.89 0.30 0.48 0.27 0.79 0.85 0.83 0.33 0.34 0.66 0.62 0.72

1 “+” 0.48 0.61 0.44 0.51 0.22 0.56 0.67 0.44 0.33 0.85 0.84 0.84 0.83 0.86 0.85 0.83 0.81 0.84 0.91 0.85 0.86 0.86 0.85 0.85 0.51 0.44 0.49 0.86 0.91 0.32 0.52 0.23 0.75 0.85 0.80 0.31 0.30 0.75 0.72 0.80 Mock Ad5 HSV VACV 2 0.48 0.59 0.39 0.49 0.18 0.56 0.65 0.44 0.31 0.86 0.85 0.82 0.84 0.85 0.84 0.83 0.83 0.85 0.85 0.84 0.85 0.89 0.88 0.87 0.50 0.43 0.49 0.89 0.91 0.31 0.50 0.27 0.79 0.88 0.84 0.28 0.30 0.72 0.68 0.78 3

0.70 0.67 0.82 0.69 0.68 0.58 0.59 0.74 0.75 0.23 0.30 0.27 0.34 0.25 0.34 0.30 0.26 0.27 0.34 0.32 0.34 0.27 0.28 0.27 0.71 0.69 0.58 0.30 0.32 0.31 0.68 0.75 0.22 0.34 0.32 0.79 0.70 0.33 0.38 0.37 1

“

- ” 0.75 0.80 0.76 0.76 0.68 0.78 0.86 0.81 0.76 0.43 0.49 0.49 0.51 0.46 0.51 0.47 0.45 0.45 0.53 0.56 0.55 0.47 0.50 0.51 0.70 0.81 0.77 0.48 0.52 0.50 0.68 0.65 0.38 0.56 0.48 0.75 0.71 0.50 0.58 0.58 2 HSV 0.55 0.56 0.65 0.68 0.72 0.55 0.55 0.68 0.84 0.21 0.26 0.23 0.31 0.22 0.28 0.25 0.20 0.20 0.28 0.33 0.34 0.24 0.26 0.28 0.66 0.63 0.57 0.27 0.23 0.27 0.75 0.65 0.19 0.30 0.33 0.69 0.73 0.23 0.31 0.28 3 0.44 0.45 0.33 0.41 0.16 0.42 0.41 0.39 0.27 0.79 0.76 0.74 0.73 0.76 0.73 0.77 0.79 0.77 0.72 0.71 0.75 0.77 0.79 0.78 0.46 0.37 0.38 0.79 0.75 0.79 0.22 0.38 0.19 0.83 0.84 0.27 0.21 0.49 Mitochondrion0.46 0.53 1 “+” Unique identifications 0.57 0.60 0.45 0.53 0.29 0.59 0.65 0.50 0.39 0.85 0.84 0.81 0.82 0.84 0.83 0.83 0.82 0.82 0.83 0.84 0.87 0.87 0.90 0.88 0.52 0.50 0.54 0.85 0.85 0.88 0.34 0.56 0.30 0.83 0.89 0.34 0.35 0.65 0.63organization0.74 2 2500 0.50 0.51 0.37 0.53 0.25 0.50 0.51 0.46 0.37 0.80 0.79 0.75 0.78 0.77 0.77 0.76 0.77 0.74 0.77 0.78 0.81 0.80 0.82 0.85 0.54 0.44 0.48 0.83 0.80 0.84 0.32 0.48 0.33 0.84 0.89 0.30 0.31 0.59 0.53 0.64 3 Viral proteins (161)

0.68 0.76 0.77 0.63 0.74 0.63 0.64 0.74 0.80 0.20 0.30 0.31 0.30 0.23 0.30 0.30 0.26 0.23 0.30 0.32 0.33 0.25 0.23 0.30 0.62 0.77 0.66 0.33 0.31 0.28 0.79 0.75 0.69 0.27 0.34 0.30 0.74 0.25 0.40 0.34 “ VACV 1 - ” 2000 0.60 0.63 0.64 0.64 0.82 0.62 0.64 0.70 0.83 0.18 0.27 0.27 0.29 0.26 0.31 0.32 0.19 0.22 0.33 0.35 0.37 0.23 0.28 0.32 0.63 0.74 0.67 0.34 0.30 0.30 0.70 0.71 0.73 0.21 0.35 0.31 0.74 0.28 0.43 0.40 2 0.47 0.63 0.43 0.52 0.20 0.54 0.68 0.42 0.27 0.68 0.69 0.69 0.69 0.73 0.75 0.69 0.65 0.74 0.76 0.72 0.72 0.71 0.69 0.69 0.51 0.42 0.49 0.66 0.75 0.72 0.33 0.50 0.23 0.49 0.65 0.59 0.25 0.28 0.85 0.89 1 1600 “+” 1500 0.59 0.67 0.57 0.57 0.33 0.62 0.72 0.55 0.40 0.63 0.63 0.66Ʃ=1,5020.64 0.66 0.70 proteins0.64 0.60 0.69 0.74 0.69 0.66 0.66 0.65 0.64 0.57 0.54 0.60 0.62 0.72 0.68 0.38 0.58 0.31 0.46 0.63 0.53 0.40 0.43 0.85 0.85 2 1400 0.55 0.67 0.47 0.58 0.28 0.60 0.75 0.50 0.37 0.74 0.76 0.75 0.76 0.78 0.79 0.74 0.69 0.77 0.82 0.80 0.80 0.78 0.76 0.76 0.58 0.48 0.54 0.72 0.80 0.78 0.37 0.58 0.28 0.53 0.74 0.64 0.34 0.40 0.89 0.85 3 Oxidation-reduction Cellular host proteins (2,170) 1200 1000 process

1000 proteins Numberof only in background 500 800 t-test p-value >0.05 t-test p-value <0.05 0 600 Acetyl-CoA metabolic

process

Total 147 proteins 147

Number of proteins Numberof 400

Ad_02 Ad_03

Carboxylic acid metabolic Ad_01

Host_01 Host_02 Host_03 Host_04 Host_05 Host_06 Host_07 Host_08 Host_09 Host_10 Host_11 Host_12 Host_13 Host_14 Host_15 HSV_01 HSV_02 HSV_03

Ad_Total

VACV_02 VACV_03

200 process VACV_01 HSV_Total

Ʃ=147 proteins Host_Total VACV_Total 0

Cellular metabolic process/cell adhesion/cell Total host Total host Total host morphogenesis proteins proteins proteins 2,123 1,808 1,770

-5.79 -0.27 log2 fold change Total host (“+” vs “-” biotin) proteins 1,636 t-test p-value > 0.05, or not detected Statistical differences Ad5 HSV VACV 45 FIP1L1 CIRH1A COPA COPB1 MCM4 POLR1B DDX27 CSTF3 ILF3 MTHFD1 TUBB2C SAFB2 GNL3 NAT10 HNRNPUL1 FLNA VPS35 RRP9 CPSF7 PRPF4 POLDIP3 EEF1A1 HSPA1A SLTM MTHFD1 SYMPK HNRNPC MYH14 LASP1 40 SMU1 GARS SF3A3 ALYREF CYFIP1 DYNC1LI1 SF3B1 ATM QARS PFKL DDX18 SRSF7 POLR1B DHX15 PDLIM7 MAGEC1 ZNF326 SRSF1 FBL ILF2 COPG1 BAG2 EEF2 ENO1 CPSF7 NCBP1 CCT6A HK1 LIG3 35 GSTP1 TRA2A DDX39B PDCD11 DPYSL2 TUBB NAT10 SSB SF3A2 POP1 EXOC4 TUBA1B POLA2 MCM5 TRIM28 RBMX EIF5B PRPF40A HNRNPH1 PDS5A CCT7 MYO1B SFRS3 LDHB RALY NUDT21 IARS HSPA8 GTF2I BAZ1B TIMELESS SF3B3 NOL6 AP3B1 DYNC1H1 PRPF19 CPSF1 PCNA POLR2A GTF2I DDX5 HNRPK VARS MARS 30 RAD50 DDX39B NONO U2AF2 SRRM2 SRRT AP3D1 STRAP SFPQ OLA1 SNRNP200 SF3B14 EPRS HOOK3 CLSPN CLSPN WIZ SUPT16H MRE11A DNMT1 MYBBP1A COPE RPS24 RFC2 SBSN WDR33 GLI2 ERC1 RFC1 PDCD11 RRM2 RFC3 LTA4H NAT10 GLI3 TUBGCP2 ATAD2 POLE EBNA1BP2 ESF1 POLR1A TCOF1 ERCC6L NCAPD2 25 GPATCH4 CHTOP EHMT2 NOP2 CIRH1A TTF2 TOP2B

value TIMELESS

- NACC1 TPX2 PNO1 NOLC1 EBNA1BP2 CSTF1 SLAIN2 KNTC1 p NOC2L KPNA4 WTAP PERP USP9X CKAP5 POLA1 C7orf26 NOL11 RCL1 FIP1L1 COPG2 EIF4A2

test ATAD5 - t 20 WDR75 AKAP17A AKAP8 HNRNPK TTC27 KIF11

2 ZNF644 RCL1 NRD1 RRP12 SGCD SPAG5 SMC4 ZNF22

log NOP2 HSSG1 RELA FHL2 CORO7 PPP1R13L - KRT121P RRP12 GMFB POP1 CAMK2D PFKL CCNB1 TCOF1 PDCD11 FBL DTX3L COPB2 15 DDX21 TBL3 CDCA7L SEMG2 PRPF4 NCAPH FAM111B SFRS18 PLP1 GNL3 BOP1 PDLIM7 PPP1R12A LTF HBA1 USP15 CSTF3 SCYL1 AP3D1 HEATR1 PCDHB4 EGR1 CSTF2 CCNB1 EXOC4 RSL1D1 NOL6 WDR43 EXOSC3 RRM1 DYNC1LI1 10 CIRH1A URB2 NOL6 NOL11 MYH10 MYH9

5

0 -10 -5 0 5 10 -15 -10 -5 0 5 10 15 -10 -5 0 5 10

log2 fold change (virus vs mock)

Artwork of Katarzyna Kulej

Page 5 Identification and quantification of selected gel bands (in-gel digestion)

Ingredients (reagents)

- HPLC grade acetonitrile - 100 mM ammonium bicarbonate, pH 8.0 - Dithiothreitol concentrated stock solution (e.g. 1 M) - Iodoacetamide, powder - Trypsin sequencing grade (e.g. Promega, catalog # V5111) - 5% formic acid in HPLC grade water

Recipe (protocol)

Estimated preparation time: 4 hours + overnight trypsin digestion - Cut around the protein band(s). Cut the excised piece into roughly 1 mm3 cubes and transfer them to a clean 1.5 mL or 0.5 mL tube. Note: If you cannot see even a very pale band after Coomassie staining, it is unlikely that you will have good results in the analysis - Wash the gel pieces with 5 times gel volume using water (~ 100 μL) 10 min on a shaker. Remove and add 50% acetonitrile 10 min. Repeat the operation twice, then shrink with 100% acetonitrile ( ~ 50 μL)

- Swell the gel particles in 10 mM dithiotreitol/0.1 M NH4HCO3 ( ~ 50 μL) and incubate for 45 min at 56°C. Chill tubes to room temperature. Remove excess liquid and replace it quickly with roughly the same volume of iodoacetamide solution (10 mg/ml (55 mM) iodoacetamide in 0.1 M NH4HCO3). Incubate for 30 minutes in the dark at room temperature - Wash the gel pieces with 5 times gel volume using water (~ 100 μL) 5 min on a shaker. Remove and add 50% acetonitrile 5 min. Repeat the operation twice, then shrink with 100% acetonitrile ( ~ 50 μL) - On ice (4°C) rehydrate gel particles in 50 mM NH4HCO3 and 12.5 ng/μL of trypsin. Add enough digestion buffer to cover the gel pieces. (~ 10 μL). Add more buffer if all the initially added volume has been absorbed by the gel pieces. After 45 min remove remaining supernatant and replace it with 5-20 μL of 50 mM NH4HCO3 without trypsin (37°C, overnight) - Transfer supernatant with peptides to a clean 1.5 mL tube. Extract more peptides from the gel by addition enough to cover the gel pieces: 5% formic acid - 15 min (~ 20 μL). Add the same volume of acetonitrile - 15 min. (~ 20 μL). Recover the supernatant. Repeat twice and dry the recovered supernatant in a vacuum centrifuge

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. A single gel band is a relatively simple mixture, which can run with a gradient of about 1 hour. This implies that in one day you can fit about 15-20 samples * All figures with in-gel and in-solution digestion are interchangeable

unique Ad5

15 translation 1 - MED30 BEND3 initiation elongation POLR3C ZHX1 10 Host proteins super- NAP1L4 MAGEC1

MS/HSV enriched in Ad5 - SARS AATF 5 NSF TTC27 DLGAP5

0 termination 0 500 1000 1500 unique HSV proteins -5 ORC2 SEPHS1 tRNA -10 Host proteins super- KIAA0101 fold change Ad5_iPOND(+) foldchange aminoacylation

2 enriched in HSV-1 EMSY

translation regulation for protein log -15 translation

mRNA processing chromatin modification

RNA export

histone rRNA processing modification mRNA splicing

transcription regulation

maturation of 5.8S rRNA response to DNA damage histone modification

mRNA metabolic process DNA metabolic process response to DNA replication DNA damage G2/M transition

0.4 4.9 -0.3 -5.3

log2 fold change Ad5/HSV

Comparison with existing datasets

Artwork of Katarzyna Kulej

Page 7 Identification and quantification of proteins in detergents, e.g. immunoprecipitations

Ingredients (reagents)

- S-traps (ProtiFi, https://www.protifi.com/s-trap). Use the “micro” version for <100 µg of proteins - 12% phosphoric acid - Loading buffer: 90% methanol, 10 mM ammonium bicarbonate, pH 8.0 - Dithiothreitol concentrated stock solution (e.g. 1 M) - Iodoacetamide, powder - Trypsin sequencing grade (e.g. Promega, catalog # V5111) + 50 mM ammonium bicarbonate - 0.2% formic acid in HPLC grade water

Recipe (protocol)

The protocol is intended for S-trap micro. For larger S-traps, adjust volumes as described in the ProtiFi website (https://www.protifi.com/s-trap) - Reduce and alkylate proteins as in Page 4 (5 mM DTT for 1 hour, followed by 20 mM iodoacetamide or 30 min in the dark). The ideal final volume is 25 µL - Add to the sample ~12% aqueous phosphoric acid at 1:10 for a final concentration of ~1.2% - Add 165 µL of Loading buffer to the sample - Load the sample into the S-Trap micro. Use an Eppendorf tube to hold the S-trap - Centrifuge gently, until all the volume passed through, e.g. 500 g for 30 sec. Repeat the load if you had a bigger sample volume - Wash twice with 150 µL of Loading buffer. Centrifuge gently at each wash - Move the S-Trap into a clean Eppendorf tube for the digestion - Add 20 µL of trypsin at the concentration of 0.1 µg/µL in 50 mM ammonium bicarbonate. Leave the digestion at least 1 hr at 47°C. Do not shake - Centrifuge gently - Elute peptides with 40 µL of 0.2% aqueous formic acid - Perform a second elution with 35 µL of 50% acetonitrile containing 0.2% formic acid. Pool elutions - Dry eluted peptides

Estimated costs

Reagents are all cost effective, but the S-Trap cartridges need to be purchased for SDS-containing samples. The price is about $250 for 40 preparations. The Core charges per time. An immunoprecipitation is a relatively simple mixture, which can run with a gradient of about 1 hour. This implies that in one day you can fit about 15-20 samples Ad HSV VACV 35.0 35.0 35.0

30.0 30.0 30.0

25.0 25.0 25.0

Enrichment

20.0 Fold change vs control (x-axis)20.0 and protein abundance (y-axis) 20.0 value - Potential pro-viral

cellular factors

test p test

-

t

2 log

- 15.0 15.0 15.0 Ad HSV VACV 35.0 35.0 35.0 10.0 10.0 10.0

Potential anti-viral p-value <0.05 cellular factors p-value >0.05

30.0 30.0 30.0 5.0 5.0 Ad 5.0 HSV VACV 35.0 35.0 35.0

25.0 0.0 25.0 0.0 Reyes et al. Mol 25.0Cell0.0 Proteomics (2017) -10.0 -5.0 0.0 5.0 10.0 -10.0 -5.0 0.0 5.0 10.0 -10.0 -5.0 0.0 5.0 10.0 log fold change (viral_iPOND(+)-MS/host_iPOND(+)-MS) 30.0 2 30.0 30.0 vesicle mediated transport chaperon mediated protein folding protein folding chromosome condensation

20.0 20.0 20.0

Ad HSV VACV value

- 35.0 35.0 35.0

25.0 25.0 25.0

test p test

- t

2 30.0 30.0 30.0 log

- 15.0 15.0 15.0

25.0 25.0 25.0

20.0 20.0 20.0

value - 20.0 20.0 20.0

value 10.0 10.0

10.0 -

test p test

test p test

-

-

t

t

2

2 log

- 15.0 15.0 15.0 log

- 15.0 15.0 15.0

5.0 10.0 5.0 10.0 10.0 5.0

5.0 5.0 5.0 10.0 10.0 10.0

0.0 0.0 0.0 0.0 0.0 0.0 -10.0 -5.0 0.0 5.0 -10.0 10.0-5.0 0.0-10.0 5.0 10.0-5.0 -10.0 -5.0 0.0 0.0 5.05.0 10.0 -10.0 10.0 -5.0 -10.00.0 5.0 -5.0 10.0 0.0 5.0 10.0 log2 fold change (viral_iPOND(+)-MS/host_iPOND(+)-MS)

logvesicle2 fold mediated change transport (viral_iPONDchaperon mediated protein(+)- foldingMS/host_iPONDprotein folding (+)chromosome-MS) condensation

5.0 5.0 5.0 mRNA metabolic process tRNA metabolic process rRNA metabolic process snRNA metabolic process Artwork of Katarzyna Kulej

0.0 0.0 0.0 -10.0 -5.0 0.0 5.0 10.0 -10.0 -5.0 0.0 5.0 10.0 -10.0 -5.0 0.0 5.0 10.0

log2 fold change (viral_iPOND(+)-MS/host_iPOND(+)-MS) Page 9 mRNA metabolic process tRNA metabolic process rRNA metabolic process snRNA metabolic process Time-resolved proteomics (multiple time points to be analyzed as regulation profiles)

Ingredients (reagents)

- HPLC grade acetonitrile - 100 mM triethylammonium bicarbonate, pH 8.0 - Dithiothreitol concentrated stock solution (e.g. 1 M) - Iodoacetamide, powder - Trypsin sequencing grade (e.g. Promega, catalog # V5111) - Tandem Mass Tag (TMT) kit (Thermo, catalog # 90061 for 6-plex or # 90406 for 10-plex)

Recipe (protocol)

Please, follow the link below for the detailed protocol - The protocol is very similar to the canonical in solution digestion described in Page 4. After protein digestion, the different samples are labeled using the TMT kit, and then mixed together to run them as a single sample. This reduces cost and missing values between time points, because peptides need to be identified just once to obtain quantification for all the mixed conditions http://www.biotech.cornell.edu/sites/default/files/uploads/Documents/Proteomics_protocols/Protoc ol%2017_TMT%20labeling.pdf

Very important! At the stage of peptide labeling with the TMT kit, you cannot utilize any buffer containing free amine groups. This includes: - Ammonium bicarbonate (use triethylammonium bicarbonate instead) - Tris - Urea - RIPA If urea and/or RIPA buffers are needed for e.g. to lyse cells, it is fundamental to desalt the peptides prior labeling. Use for example the C18 cartridges WAT023501 (Waters)

Estimated costs

All reagents adopted in this protocol are cost effective and likely present in other labs. The TMT kit is the most expensive; it can vary between $600 and $1,100 depending if you want 6 or 10 time points Please, consider that the TMT kit can be aliquoted, but it is not stable for long time once resuspended. Also, consider that all time points are analyzed as a single sample, reducing the overall Core service cost Commercial description of the method (Thermo Scientific)

Time course of HSV-1 infection Time course of HSV-1 infection Cluster Number Cluster number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Process mRNA processing mRNA splicing, via spliceosome P ncRNA processing positive regulation of RNA biosynthetic process regulation of mRNA processing regulation of mRNA stability regulation of RNA splicing RNA localization RNA splicing rRNA processing DNA packaging regulation of DNA replication DNA conformation change translation translational initiation translational termination ribosome biogenesis

RNA metabolic process metabolic RNA gene expression regulation of gene expression, epigenetic positive regulation of gene expression negative regulation of gene expression posttranscriptional regulation of gene expression chromosome organization

chromatin organization DNA metabolic process metabolic DNA chromatin modification chromatin assembly or disassembly

Gene expression Gene histone modification cell cycle cell cycle G2/M phase transition cell cycle arrest protein transport vesicle-mediated transport cytoskeleton-dependent intracellular transport

Cell cycle Cell Golgi vesicle transport nuclear transport RNA transport nucleocytoplasmic transport Chromatin organization Chromatin regulation of cytoplasmic transport cytosolic transport ER to Golgi vesicle-mediated transport exocytosis nuclear import mRNA export from nucleus nuclear export protein localization to plasma membrane protein localization to organelle antigen processing and presentation immune response apoptotic process

Intracellular transportIntracellular positive regulation of apoptotic process negative regulation of apoptotic process programmed cell death positive regulation of cell death negative regulation of cell death cellular response to stress

Immunesystem cellular response to DNA damage stimulus DNA repair

Cell death Cell cytoskeleton organization actin cytoskeleton organization microtubule cytoskeleton organization negative regulation of cytoskeleton organization actin filament-based movement actin filament organization microtubule cytoskeleton organization phosphorylation dephosphorylation

Other mitochondrion organization mitochondrial translation protein localization to mitochondrion nucleotide metabolic process pyridine nucleotide metabolic process 1.29E-22 9.98E-01 GO p-value enrichment

Kulej, Augusti, Sidoli et al. – Molecular and Cellular Proteomics (2017)

Nicetto et al. Science (2019) – Example with RNA-seq data

Page 11 Comprehensive quantification of histone post-translational modifications

Ingredients (reagents)

- HPLC grade acetonitrile - 100 mM ammonium bicarbonate, pH 8.0 - Propionic anhydride - Ammonium hydroxide - Glacial acetic acid - Trypsin sequencing grade (e.g. Promega, catalog # V5111)

Recipe (protocol)

Estimated preparation time: 1 hour + overnight digestion + 1 hour. The protocol is designed for multi-channel pipettes to process many samples. Obviously, it can also be performed with single Eppendorf tubes

1. Resuspend at least 20 μg of histones in 20 μL of 50 mM ammonium bicarbonate (NH4HCO3), pH 8.0. The amount of material does not need to be precise, but do not exceed 100 μg. 2. Place each sample in a well of a 96-well plate (e.g. link). Mark in an excel table which sample is in which well. 3. Add 5 μL of acetonitrile in each well using a multi-channel pipette (8 or 12 channels). Solutions can be poured into a polypropylene reservoir (e.g. link). 4. (Under the hood) Propionylate histones by adding 2 μL of propionic anhydride (e.g. link) rapidly

followed by 9 or 10 μL of ammonium hydroxide (NH4OH). Pipette up and down a few times just with the NH4OH pipette to mix the reaction. 5. By using a multi-channel pipette, dip into one row or one column with P10 pipette tips (no need to

aspirate). Touch with the tips a pH indicator strip. NH4OH and glacial acetic acid can be used to adjust the pH to around 8.0. If the result seems ambiguous (different pH across samples), test more rows/columns and adjust the pH. 6. Wait 10-15 min. 7. Repeat steps 4-6 (step 4 under the hood). 8. Dry plate(s) in a SpeedVac centrifuge. They will likely not dry completely, because of the viscosity of the propionic anhydride. There is no need to leave them more than 2 hours.

9. Resuspend trypsin (e.g. link) to a concentration of 25 ng/μL in 50 mM NH4HCO3 and pour it into the reservoir. Add 30 μL of solution in each sample with the multi-channel pipette. Pipette up and down a few times to eliminate the viscosity of the residual liquid in the bottom of the wells. 10. Check the pH as step 5. This is important! In acidic pH trypsin is not active. For extra safety, it is suggested to check the pH again after e.g. 1 hour of trypsin incubation. 11. Wait overnight or at least 6 hours. 12. Repeat steps 3-8. 100% (H3 aa 18-26) KQLATKAAR 1ac 90% 80% 70% 60% 50% 40%

30% H3K18ac MS/MS Relative abundance Relative 20% 10% 0%

H3K23ac MS/MS

0.8 y = 0.95x + 0.02 0.6 0.4 Sidoli et al. – Analytical Chemistry (2017) BUday 0 0.2 0.0 0.0 0.2 0.4 0.6 0.8 MD day 0

25.00%

4day RA A 4day ESCs A ESCs B ESCs A EBs B EBs RA A 1day RA B 1day RA B 4day RA A 7day RA B 7day H3.3K36me1 K36me1 H3K36me3 20.00% H4K20me2 K36me2 H3.3K36me3 H4K20me3 K36me3 H3K14ac 15.00% H3K36me2 K36ac H4K16ac K36me1:1 H3.3K27me2 H3K9me3 K36me2:1 H3.3K36me2 10.00% H3K27me1 K36me2:2 H3K9me2 H3K79ac K36me3:1 H3K18me1 5.00% K36me3:2 H3K23me1 H3.3K27me1 K36me3:3 H4K20me1 H4K20ac H3K4ac 0.00% H3K122ac Day 0 Day 1 Day 2 H3T22ac H3K4me3 H3K4me2 H4K12ac H4K8ac Sidoli et al. – Epigenetics & Chromatin (2017) H4K5ac H3K23ac H3K18ac H3K9ac H3K79me3 H3K56ac H3K56me2 H3K56me1 H3K36me1 H3K79me1 H3.3K27me3 H3K27me3 H3K27me2 H3K79me2 H3K9me1 H3K4me1

Z-score -2 0 2

Gonzales-Cope, Sidoli et al. – BMC Genomics (2016)

Page 13 Quantification of synthesis/degradation rate of proteomes using pulsed metabolic labeling

Ingredients (reagents)

- Media for cell growth requires additional lysine and arginine with stable isotopes (13C and 15N) - All the other reagents are the same as Page 4

Recipe (protocol)

Follow the same protocol as Page 4 This procedure provides a complementary quantification dimension to protein abundance, which is the turnover rate of the protein synthesis/degradation. A protein might not change much its abundance between two conditions, but it might be expressed and degraded faster/slower. To obtain this information, proteins should be only “partially labeled”, meaning that a complete incorporation of labeled amino acids should not be achieved. To obtain this effect, we recommend labeling proteins for an interval of time corresponding to 1-2 cell cycles

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. A single gel band is a relatively simple mixture, which can run with a gradient of about 1 hour. This implies that in one day you can fit about 20 samples Heavy arginine and lysine are more expensive than the canonical ones. The cost is highly dependable on the volume where the cells are grown into. Consider the amount of material required to minimize the costs Replace light media with heavy media

(-k * t) Fh(t) = 1 – e Rate = 0.020 hr-1 Half life = 34.4 hr

Page 15 Protein – RNA interaction analysis to identify nucleic acid binding domains of proteins

Ingredients (reagents)

- Canonical proteomics reagents (Page 4) - 4-Thiouridine (4sU), e.g. Sigma-Aldrich catalog # T4509 - UV light - Benzonase or RNAse A to digest RNA on peptides - Cell culture to incorporate 4sU in the nascent RNA

Recipe (protocol)

Please, follow the protocol described in this manuscript: https://www.jove.com/video/56004/sample-preparation-for-mass-spectrometry-based- identification-rna

An application of this workflow can be found at: https://www.ncbi.nlm.nih.gov/pubmed/27768875

The Proteomics Core will then assist you with data processing, statistics and interpretation

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 4-thiouridine is about $170 for 25 mg The cost of a UV-B lamb can be below $1,000 Protocol (RBP = RNA binding protein)

Warneford-Thomson, He, Sidoli et al. JoVE (2017)

He, Sidoli et al. Molecular Cell (2016)

Page 17 Identification of protein complexes associated on the chromatin (ChIP-SICAP)

Ingredients (reagents)

- Canonical proteomics reagents (Page 4) - Other reagents described in the link

Recipe (protocol)

This protocol allows for the identification of protein-protein interactions specifically on chromatin. The protocol can be found at the following publication: https://www.cell.com/molecular-cell/pdfExtended/S1097-2765(16)30568-8

Please, consider a learning curve and optimization for this procedure. The Proteomics Core is glad to assist with guidance and edits to the protocol.

Estimated costs

N/A Rafiee et al. – Molecular Cell (2016)

Page 19 Estimation of the turnover rate of post- translational modifications

Ingredients (reagents)

- Please, see next page for the list of metabolites that can be used to label newly synthesized post- translational modifications - The rest of the reagents are the same as described at Page 4

Recipe (protocol)

Follow the same protocol as Page 4 This procedure provides a complementary quantification dimension to the abundance of post- translational modifications, which is their turnover rate. A modification might not change much its abundance between two conditions, but it might be catalyzed and removed faster/slower. To obtain this information, modifications should be only “partially labeled”. Please, see next page to have an approximate idea about the catalysis speed of different modifications.

To obtain a real complete estimation of the turnover rate of a modification, it is necessary to perform a normalization by the turnover rate of the protein itself. For estimating the turnover rate of a protein, please see the protocol at Page 14

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 ac acetylation acetyl-CoA min – hours

S-adenosylmethionine me methylation hours – days (SAM)

ph phosphorylation ATP sec – min

Ar ADP-ribosylation ADP ribose ???

Og O-GlcNAc UDP-N-Acetylglucosamine hours – days

suc/pr/bu/cr other acylations * acyl-CoA min – hours? hib/bhb/ma/glu (succinylation, propionylation, * X butyrylation, crotonylation, hydroxyisobutyrylation, β-hydroxybutyrylation, malonylation, glutarylation) Simithy, Sidoli and Garcia – Proteomics (2018)

13 CD3-Methionine me (stimulation) me* Cell culture

Old PTMs New PTMs

me me me* me me* me* me* me* me me me me*

me3 me3:1 me3:2 me3:3

Sidoli et al. – Epigenetics & Chromatin (2017)

Page 21 Phosphoproteomics

Ingredients (reagents)

- Loading buffer: 80% Acetonitrile, 5% TFA and 1 M Glycolic acid (76 mg/mL) - Washing buffer 1: 80% Acetonitrile, 1% TFA - Washing buffer 2: 20% Acetonitrile, 0.2% TFA - Elution buffer: 40 µL Ammonia solution (28%) in 980 µL H2O, pH 11,3 - Titanium Dioxide (TiO2) beads, e.g. GL Sciences catalog # GL-5010-21315

Important reminder

Please, ensure that cell lysis and follow-up steps up to trypsin digestion are performed on ice (when possible) and with the presence of phosphatases inhibitors (e.g. Thermo catalog # 78426)

Recipe (protocol)

Follow the same protocol as Page 4 to obtain peptides from your cells. Then: - Dilute your peptide solution at least 10 times with loading buffer (alternatively if you have 100 µL sample you can add 50 µl water, 50 µL 100% TFA, 800 µL Acetonitrile and 76 mg Glycolic acid to make the sample up to the proper loading buffer) - Add 0.6 mg TiO2 beads per 100 µg peptide solution - Place the tubes on the shaker (highest shaking) at room temperature for 5-10 min - Centrifuge to pellet the beads (table centrifuge <15 sec) - Remove the supernatant - Wash the beads with 70-100 µL loading buffer - mix for 15 sec, transfer to another eppendorff tube and then centrifuge to pellet the beads (the transfer is performed due to the fact that peptides and phosphopeptides stick to surfaces and can be eluted in the last elution step) - Wash with 70-100 µL washing buffer 1 - mix for 15 sec and then centrifuge to pellet the beads. Remove supernatant. - Wash with 50-100 µL washing buffer 2 - mix for 15 sec and then centrifuge to pellet the beads. This step is important to remove peptides that bind in a HILIC mode to TiO2. Remove supernatant. - Dry the beads for 5-10 min in the vacuum centrifuge or on the table. - Elute the phosphopeptides with 50 µL Elution buffer – mix well and leave the solution and the beads for 5-10 min to allow an efficient elution - Centrifuge the solution for 1 min, recover the supernatant (as it contains the phosphopeptides) and dry it in a SpeedVac centrifuge PhosphoPath: Raaijmakers et al. J. Proteome Research (2015)

Kinome Render: Chartier et al. PeerJ (2013)

Motif-X: O’Shea et al. Nature Methods (2013)

Page 23