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In solution digestion

In solution digestion is performed in order to analyse all the proteins that are present in the sample. The method usually doesn’t require any protein separation. The experiment is performed in whole proteome which is to be analyzed. In solution digestion.

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In solution digestion

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  1. In solution digestion is performed in order to analyse all the proteins that are present in the sample. The method usually doesn’t require any protein separation. The experiment is performed in whole proteome which is to be analyzed In solution digestion • Related LOs: Ziptipping, Trypsin properties >> Prior Viewing – IDD-6. Extraction of serum protein, IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE, IDD-19. Coomassie staining, IDD-26. Spot picking > Future Viewing – IDD-29. Matrix preparation for MALDI analysis, IDD-31. MALDI-TOF data analysis • Course Name: In solution Digestion • Level(UG/PG): UG • Author(s): Dinesh Raghu, Vinayak Pachapur • Mentor: Dr. Sanjeeva Srivastava *The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license

  2. Learning objectives 1 After interacting with this learning object, the learner will be able to: • Define the destaining of the selected spots • Recognize to reduce and alkylate the proteins using chemical reagents • Relate the digestion of peptides using trypsin • Plan the protein extraction step the gel for mass spectrometry analysis • Interpret the results of the experiment • Assess the troubleshooting steps involved in the experiments. 2 3 4 5

  3. Master Layout 1 Reagent Preparation (Slide: 5-12) 2 Sample processing (Slide: 13-16) Reduction and alkylation (Slide: 18-19) 3 Trypsin digestion (Slide: 20-21) 4 Stopping the reaction (Slide: 22) Storage (Slide: 23) 5 Display a image from each of these steps, with user click.

  4. Definitions and Keywords 1 Reduction solution (digestion buffer ): It consists of dithiothreitol and 25mM of ammonium bicarbonate. Reduction solution denatures the protein to its primary structure by reducing the disulphide bond. Alkylation solution: It consists of iodoacetamide which prevents the reformation of disulfide bond. Trypsin: A proteolytic enzyme that is used for digestion of large proteins into smaller peptides . The digestion takes place at the carboxy terminal end of basic amino acids like arginine and lysine. 2 3 4 5

  5. Step 1: T1: Reagents preparation 1 Water Tris-HCl 2 Tris-Hcl PH 8 3 Audio Narration Description of the action Animator should redraw above figure as shown. Instruct user to weigh Tris-HCl, user must press tare in the balance with paper to show reading 0.00g, the user should click on the spatula open the lid of the Tris-HCl bottle and weigh 1.2g . In case if the gram exceeds user should remove some quantity or if it is low add to get required amount. Transfer the weighed amount to Tris-HCl solution bottle, now instruct user to pour water in the measuring cylinder till the level of water reaches to 8ml. Later transfer the solution into the Tris-HCl solution bottle and show like mixing as shown in slide-6, followed by pouring in the beaker and checking pH The recipe is standard for carrying out the In-solution digestion experiment. 4 5

  6. Step 1: T1: Reagents preparation 1 2 Beaker Magnetic bead 3 Description of the action Audio Narration (if any)‏ Show magnetic stirrer instrument. Let user place the beaker on it. Display the beaker containing powder at bottom, liquid layer on top and a magnetic bead at the bottom. Instruct user to ON the instrument, let user control the speed nob and regulate it accordingly to control the mixing speed in the beaker. Animate powder getting into the solution. Show a turbid solution turning colorless 4 The magnetic stirrer helps for the evenly distribution of the solute into the solution. 5 Video file: Magnetic stirrer

  7. Step 1: T1: Reagents preparation 1 2 NaOH 3 HCl Audio Narration Description of the action Then the bottle containing(labeled as “Tris-HCl pH8”) has to be taken near pH meter and allow the user to dip pH rod in the solution. Animate like the user switching on the pH meter. The meter should show 4.2-5.0 in the display and instruct user to add NaOH. Now allow the user to click on NaOH so that drops of NaOH should be added using filler and the reading should increase like 4.2, 4.6, 4.8, 5.2, 5.6, 5.8…..8 (desired pH) pour it to the measuring cylinder and add water to make the volume to 10ml in the cylinder. The user should click on hands for the things to happen. Prepare pH-8 Tris-HCl solution, the exact pH is required for better results. 4 5

  8. Step 1: T1: Reagent Preparation 1 Water ABC 2 Audio Narration Description of the action 3 Animator should redraw above figure as shown. Instruct user to weigh ABC (ammonium bicarbonate), user must tare the balance with paper to show reading 0.00g, with help of spatula weigh the required amount (0.790g) Show like the user added excess amount and show like transferring the excess to the ABC Transfer it to bottle labeled as 100mM ABC, followed by addition of water. Animate like the user taking the Milli-Q water and pouring to the measuring cylinder till the volume reaches 80ml and adding to the bottle and mixing as in slide 9 and show like transferring the solution to the measuring cylinder and click on Milli-Q water , pour in the cylinder till volume reaches 100ml ABC is used to for reducing the interactions between protein and dye. 4 5

  9. Step 1: T1: Reagent Preparation 1 Water DTT 2 Audio Narration Description of the action 3 Animator should redraw above figure as shown. Instruct user to weigh 7.5mg of DTT, user must tare the balance with paper to show reading 0.00g, with help of spatula weigh the required amount (0.075g) Show like the user added excess amount and show like transferring the excess to the DTT bottle and transfer weighed DTT to bottle labeled as ”Reduction solution”, followed by addition of 1ml of 100mM ABC. Animate like the user taking the pipette ,setting the value to 1000ul ,pipetting out 100mM ABC and pouring to the weighed DTT and adding to the bottle and mixing like the user shaking the tube. Prepare 100mM DTT using 100mM ammonium bicarbonate (ABC) and mix them well. 4 5

  10. Step 1: T1: Reagent Preparation 1 Water IAA 2 Audio Narration Description of the action 3 Prepare 100mM Iodoacetamide (IAA) using 100mM ammonium bicarbonate and mix them well. Different reagent preparation are used for protein treatment. Animator should redraw above figure as shown. Instruct user to weigh 20mg of iodoacetamide, user must tare the balance with paper to show reading 0.00g, with help of spatula weigh the required amount (0.020g) Show like the user added excess amount and show like transferring the excess to the IAA bottle and Transfer weighed IAA to bottle labeled as “Alkylating solution”, followed by addition of 1ml of 100mM ABC. Animate like the user taking the pipette ,setting the value to 1000ul ,pipetting out 100mM ABC and pouring to the weighed IAA and adding to “50mM IAA“ bottle and mixing like the user shaking the tube 4 5

  11. Description of the action Audio Narration Step 1: T1: Reagent Preparation 1 Please make calculation for 0.5% acetic acid depending upon the sample size as it is used for the extraction step. Show a bottle labeled as “acetic acid” and measuring cylinder. 2 Show a measuring cylinder, the user must click on the “Milli-Q” water. Animate like the user taking the pipette, setting the value to 5ul, pipetting out 10ul of the water and adding to the tube labeled as”0.5 % acetic acid” 3 4 5

  12. Description of the action Audio Narration Step 1: T1: Reagent Preparation 1 Prepare make calculation for 5% acetonitrile depending upon the sample size as it is used to reduce the the adsorption of peptides on surface of tubes and on tips. Show a bottle labeled as “acetonitrile” and measuring cylinder. Animate like the user taking the pipette, setting the value to 500ul, pipetting out the acetonitrile (when the user clicks on it) and adding it to the bottle labeled as”5 % acetonitrile”. Show a measuring cylinder, the user must click on the “Milli-Q” water and pour till the volume reaches 9.5 ml and show like adding the water to the bottle labeled as”5% acetonitrile. 2 3 4 5

  13. Description of the action/ interactivity Audio Narration (if any)‏ Step 2: T2:Sample Processing 1 2 3 Animate to take out the sample from -20’c freezer by opening the freezer. The solution inside the tube must look like frozen. Now instruct user to thaw the tube, by holding tube between palms with rubbing action (action should happen as and when the user clicks on the hand). After thawing, display the change in phase to liquid form. Show a bucket of ice and animate like placing the tube on ice. Thaw the frozen sample by rubbing between the palms. Sudden temperature change may result in harsh treatment on the sample, which may result in protein property loss. 4 5

  14. Description of the action Audio Narration Step 3: T2: Sample processing 1 Show the tube labeled as “sample” and a tube labeled as “ 5% acetonitrile and 100mM ammonium bicarbonate. Animate like the user taking the marker and label the tube as “trypsin digest”. Animate like the user taking the pipette, setting the value to 200ul, pipetting out the sample (when the user clicks on it) and adding it to the bottle labeled as trypsin digest ” Now animate user taking the pipette, setting the value to 200ul, pipetting out the acetonitrile (when the user clicks on it) and adding it to the bottle labeled as “trypsin digest. Add 100mM ammonium bicarbonate and 5% acetonitrile to the sample. This is to de-stain the sample solution as a additional step. 2 3 4 5

  15. Description of the action Audio Narration ‏ Step 3: 1 T2: Sample processing 2 Show the color as dark green than blue 3 Animate like the user taking the pipette,setting the value to 5ul, pipetting out the sample from trypsin digest tube and keep it on the paper (when the user clicks on it) as shown at one end now show gradually a change of color to dark blue 4 The pH of the sample should be 8 for better trypsin activity. 5

  16. Description of the action Audio Narration Step 3: T2: Sample processing 1 Show in the other way if the color change to green (show in a tab” acidic pH”) and the bottle labeled as”1M tris” Animate like the user taking the pipette, setting the value to 10ul, pipetting out the tris and add to the sample and show like mixing using the pipette,(when the user clicks on it) now again do the same procedure as in slide:12. If the pH is acidic , add tris –hcl bring down to pH:8 to make it “basic pH” 2 3 4 5

  17. Step 4: T3: Reduction and alkylation 1 2 60 C 3 Dry Bath 4 Please re-draw the figure. Tube labeled as reducing agent…not dehydration solution 5

  18. Description of the action Audio Narration Step 4: T3: Reduction and alkylation 1 Animate like the user taking the tube in hand and showing solution inside it as shown in slide. Switch on the dry bath when the user clicks on it and show like the user setting the temperature to 60’C by clicking on the temperature button. redraw the figure Animate like the user taking the pipette and setting the value to 50ul . The user should click on the “ reduction solution” and open the lid to pipette out 50ul of the solution and adding it to the tube with solution and keeping it in a dry bath at 60C for 5-10minutes. now the user should click on the bottle labeled as “50mM ABC” and add 50ul (set) to it and show like leaving for 2 minutes and show like the user removing the water layer after 2 minutes. Show the clock running as per time specified Add 50ul of reduction solution and keep for 5-10 minutes at 60’C , then remove the supernatant and add 50ul of 50mM ammonium bicarbonate to the tube and keep for 2 minutes, and remove the supernatant 2 3 4 5

  19. Description of the action Audio Narration Step 5: T3: Reduction and alkylation 1 Animate like the user taking the pipette and setting the value to 50ul . The user should click on the “ alkylating solution” and open the lid to pipette out 50ul of the solution and adding it to the tube with solution and keeping it in room temperature in dark (animate accordingly) for 20 minutes now the user should click on the bottle labeled as “25mM ABC” and add 50ul (set) to it and show like leaving for 2 minutes and show like the user removing the water layer after 2 minutes. Show the clock running as per time specified Add 50ul of alkylation solution and keep for 20 minutes in room temperature, then remove the supernatant and add 50ul of 25mM ammonium bicarbonate to the tube and keep for 2 minutes, and remove the supernatant. 2 3 4 5

  20. Description of the action Audio Narration ‏ Step 6: T4:Trypsin digestion 1 Show a tube labeled as “trypsin (20ug)” and a bottle labeled as 25mM ammonium bicarbonate Animate like the user taking it from -80C freezer by opening it and zoom the tube labeled as “Trypsin (20ug)” Animate like the user taking the pipette and setting the value to 1000ul and the user should click on pipette to take 1000ul of ammonium bicarbonate and show like adding to the trypsin tube . Animate like the user keeping the tube on ice Add 1 ml of 25mM ammonium bicarbonate solution to the 20ug of trypsin vial. The recipe is standard for carrying out the In-solution digestion experiment. 2 3 4 5

  21. Description of the action Audio Narration Step 7: T4:Trypsin digestion 1 Show a tube with the solution (transparent) and the 25mM ABC bottle and trypsin tube. user must take the pipette, click to set the value to 20ul (400ng) and open the trypsin tube, take the amount by clicking on the pipette and show like adding to the tube with gel solution and instruct to keep in ice. Show a clock running for 30 minutes After 30 minutes, user must take the pipette, click to set the value to 100ul and open the ABC bottle, take the amount by clicking on the pipette and show like adding to the tube with sample solution by click on the user hand and show like placing the tube in the instrument labeled as 37 C incubator, the user should open the incubator to keep the tube inside and show a clock running for 16hours. Add trypsin to the protein solution. Trypsin cleave the proteins and make it to smaller peptides. Note - Substrate: Trypsin ratio is kept 50:1 generally in most of the experiments. 2 3 4 5

  22. Description of the action Audio Narration ‏ Step 8: T5: Stopping the reaction 1 Acidify the trypsin digest mixture to cease the reaction using 0.5% acetic acid. Animate like the user taking the pipette, setting the value to 5ul, pipetting out (when the user clicks on it) from the tube labeled as”0.5 % acetic acid” and add to the tube labeled as Trypsin digest. Now again show like performing the step as in slide:12, but now color has to change to green. 2 3 4 5

  23. Description of the action Audio Narration Step 9 : T6:Sample storage 1 sample 2 3 Show like the user taking the tube and placing it in the cryo box as shown. Then animate like the user opening the -20C freezer and placing the tube inside and closing it Store the concentrated sample until further usage. Please follow the future viewing IDD for more information 4 5

  24. Button 01 Button 02 Button 03 Slide 5-12 Slide 14-16 Slide 13 Slide 17-18 Slide 19 Slide 20,21 Slide 22-23 Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07 Name of the section/stage Animation area Interactivity area Animate a question like “ If the protein extract is in the buffer , trypsin digestion can be done by Give options like “ In gel digestion and In solution digestion “ If the user clicks on the In gel digestion show a tab written as “ protocol can be used only when the protein spot is in the gel” If the user click on the In solution digestion “take him to the animation of in solution digestion” Instructions/ Working area Credits

  25. Questionnaire: APPENDIX 1 Question 1: What is the main constituent of alkylation solution? Dithiothreitol Iodoacetamide Ammonium Bicarbonate Trypsin Question 2: Reaction that occurs between Dithiothreitol and disulphide bond Alkylation reduction Iodine addition Denaturation Question 3: Trypsin is a Catalyst Inhibitor Protease enzyme Gel constituent

  26. Questionnaire: APPENDIX 1 Question 4: 0.5% acetic acid is used for Increase the pH of trypsin-sample mixture Decrease the pH of trypsin–sample mixture/to stop or reduce the activity of trypsin Neutralize the pH Stop the increase/decrease in pH Question 5: Trypsin cleave at a)Amino terminal end of acidic amino acids b) Amino terminal end of basic amino acids c) Carboxy terminal end of basic amino acids d) Carboxy terminal end of acidic amino acis

  27. APPENDIX 2 Links for further reading • Reference websites: • http://iitb.vlab.co.in • Books: • Andrew j.Link and Joshua LaBaer. “Proteomics” Cold spring harbor laboratory manual.

  28. APPENDIX 3 Summary In solution digestion involves reducing the disufide bond to denature the protein and alkylate to prevent the formation of any disulfide bond followed by protease digestion using trypsin followed by storage .

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