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In gel digestion

The proteins separated by 2D are analyzed and significant spots are excised, processed and taken for mass spectrometric analysis. Prior to the Mass spectrometry analysis it is important to cleave the protein in the gel by trypsin to make them smaller peptides for easy analysis by Mass spectrometry.

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In gel digestion

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  1. The proteins separated by 2D are analyzed and significant spots are excised, processed and taken for mass spectrometric analysis. Prior to the Mass spectrometry analysis it is important to cleave the protein in the gel by trypsin to make them smaller peptides for easy analysis by Mass spectrometry In gel 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 gel digestion • Level(UG/PG): UG • Author(s): Dinesh Raghu, Vinyak 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 for mass spectrometry analysis using Zip tips • Interpret the results of the experiment • Assess the troubleshooting steps involved in the experiments. 2 3 4 5

  3. Master Layout 1 Sample Thawing (Slide: 5-6) R Reagent preparation (Slide: 7-18) 2 Addition of stain removal solution (Slide: 19-21) Solution Treatment (Slide: 22-27) 3 Trypsin digestion (Slide: 28-29) 4 Extraction (Slide: 30-32) Zip-Tip (Slide: 33-37) Storage (Slide:38) 5 Display a image from each of these steps, with user click.

  4. Definitions and Keywords 1 1) Destaining solution: It consists of acetonitrile and 25mM ammonium bicarbonate . Acetonitrile decreases the hydrophobic interaction between the stain and the protein while the ammonium bicarbonate decreases the ionic interaction between them. 2) Reduction solution and alkylation solution: It consists of dithiothreitol and 25mM of ammonium bicarbonate. Reduction solution denatures the protein to its primary structure by reducing the disulphide bond and alkylation solution containing iodoacetamide prevent the reformation of disulfide bond. 3)Trypsin: A proteolytic enzyme that is used for digestion large proteins into smaller peptides. The digestion takes place at the carboxy terminal end of basic amino acids like aragnine and lysine. 2 3 4 5

  5. Description of the action/ interactivity Audio Narration (if any)‏ Step 1: T1: Sample Thawing 1 2 3 Animator should instruct the user to go through the IDD-26. Spot picking prior to start this experiment. User must be aware of the technique involved in spot picking and storage of excised spot. 4 5

  6. Description of the action Audio Narration Step 2: T1: Sample Thawing 1 Remove the excised spots from the 4’C and allow it thaw at room temperature. Sudden temperature change may result in harsh treatment on the sample, which may result in protein property loss. 2 Animate like the user taking the sample from the 4’C freezer (stored as in IDD-26. Spot picking) by opening and allow it thaw . Show like keeping the sample in room temperature. 3 4 5

  7. Description of the action Audio Narration Step 3: T2: Reagent preparation ‏ 1 2 3 Animator should draw the tubes, stand as in figure and animate like the user taking the marker and label the tube as “staining removal solution, dehydration solution, reduction solution, alkylation solution, extraction solution” as in figure. Label the 15ml falcon tubes as “staining solution, dehydration solution, reduction solution, alkylation solution, extraction solution” prior to reagent preparation. 4 5

  8. Description of the action Audio Narration (if any)‏ Step 3: T2: Reagent Preparation 1 2 3 Show a measuring balance. A hand action when clicked by user should ON the Instrument, pick paper from rack, place it on the balance so that balance reads 0.03g and the user should press ”0” on the balance to make the reading to “0.00” Clean the surface of the balance, Tare the weight of the paper before weighing. 4 Video file: Balancing 5

  9. Step 3: T2: Reagent Preparation 1 2 Beaker Magnetic bead 3 Description of the action Audio Narration (if any)‏ The magnetic stirrer helps for the evenly distribution of the solute into the solution. 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 knob 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 5 Video file: Magnetic stirrer

  10. Step 3: T2: Reagent Preparation 1 ABC 2 Water 3 Audio Narration Description of the action 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 (0.197g). Transfer it to empty tube labeled as 25mM 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 empty bottle for mixing as in slide:9. After mixing is done transfer the solution to the measuring cylinder and click on Milli-Q water, pour into the measuring cylinder to makeup the final volume to100ml. Weigh 0.197 g of ammonium bicarbonate. Give a brief spin to dissolve powder completely and later make the volume to 100ml. 4 5

  11. Step 3: T2: Reagent Preparation 1 ABC 2 Water 3 Audio Narration Description of the action 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 (0.395g). Transfer it to empty tube labeled as 50mM 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. After mixing is done transfer the solution to the measuring cylinder and click on Milli-Q water, pour into the measuring cylinder to makeup the final volume to100ml. Weigh 0.395 g of ammonium bicarbonate. Give a brief spin to dissolve powder completely and later make the volume to 100ml. 4 5

  12. Step 3: T2: 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 empty tube 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. After mixing is done transfer the solution to the measuring cylinder and click on Milli-Q water, pour into the measuring cylinder to makeup the final volume to100ml. Weigh 0.790 g of ammonium bicarbonate. Give a brief spin to dissolve powder completely and later make the volume to 100ml. 4 5

  13. Step 3: T2: Reagent Preparation 1 Water DTT 2 Audio Narration Description of the action 3 Animator should redraw above figure as shown. Instruct user to weigh 1.5mg of DTT, user must tare the balance with paper to show reading 0.00g, with help of spatula weigh the required amount (0.0150g). Transfer weighed DTT to empty tube 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 of ABC and pouring to the weighed DTT. User must give a brief vortex for proper mixing. Prepare 10mM DTT using 100mM ammonium bicarbonate and mix them well. 4 5

  14. Step 3: T2: Reagent Preparation 1 Water IAA 2 Audio Narration Description of the action 3 Animator should redraw above figure as shown. Instruct user to weigh 10mg of iodoacetamide, user must tare the balance with paper to show reading 0.00g, with help of spatula weigh the required amount (0.010g). Transfer weighed IAA to empty tube 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. User must give a brief vortex for proper mixing. Prepare 50mM iodoacetamide using 100mM ammonium bicarbonate and mix them well. 4 5

  15. Description of the action Audio Narration Step 3: T2: Reagent Preparation 1 Show a bottle labeled as “trifluoroacetic acid” and Milli-Q water. Animate like the user taking the pipette, setting the value to 100ul, pipetting out the Trifluoroacetic acid (when the user clicks on it) followed by adding it to the bottle labeled as”0.1 % TFA”. Draw a measuring cylinder , animate like the user clicking on the “Milli-Q bottle” and pour till the volume reaches 99ml and adding it to 0.1% TFA bottle. Animate like the user taking the pipette, setting the value to 900ul, pipetting out the water and adding it to the bottle labeled as ”0.1 % TFA” and show like inverting the bottle for mixing when the user clicks on hand. Prepare 0.1% Trifluoroacetic acid using Milli-Q water. 2 3 4 5

  16. Description of the action Audio Narration ‏ Step 3: T2: Reagent Preparation 1 Show the bottles labeled as 25mM ABC and acetonitrile and a tube labeled as “Destaining solution” (as in slide:7) Animate like the user taking the pipette and setting the value to 1000ul and pipette out 1000ul of 25mM ABC and pour in tube . Show like discard the tip and taking the new one to pipette out 1000ul of acetonitrile and add to the tube. show like mixing as shown in slide:17. when the user clicks on start button instrument must rotate like “giant wheel”. Prepare destaining solution containing 1:1 ratio of acetonitrile and 25mM ammonium bicarbonate. The recipe is standard for carrying out the In-gel digestion experiment. 2 3 4 5

  17. Step 3: T2: Reagent Preparation 1 2 3 TUBE (destaining solution) 4 5

  18. Description of the action Audio Narration ‏ Step 3: T2: Reagent Preparation 1 Show the bottles labeled as 25mM ABC and acetonitrile and a tube labeled as “Dehydration solution” (as in slide:7) Animate like the user taking the pipette and setting the value to 100ul and pipette out 100ul of 25mM ABC and pour in tube. Show like discard the tip and taking the new one to pipette out 50ul of acetonitrile, now set the pipette to 50ul to add into the tube and show like mixing as shown in slide:17. when the user clicks on start button instrument must rotate like “giant wheel”. Prepare dehydration solution containing 2:1 ratio of 25 mM ammonium bicarbonate and acetonitrile. The recipe is standard for carrying out the In-gel digestion experiment. 2 3 4 5

  19. Step 4: T3: Addition of stain removal solution 1 2 3 4 5

  20. Description of the action Audio Narration Step 4: T3: Addition of stain removal solution 1 Animate like the user taking the tube in hand and showing the blue colored pieces in inside it as shown in slide. Animate like the user taking the pipette and setting the value to 200ul. The user should click on the “stain removal solution” and open the lid to pipette out 200ul of the solution to add into the tube having blue colored gel pieces and keeping it in mixer (as in slide:17) for 30 minutes treatment. Add 200ul of destaining solution and mix the contents for 30 minutes for better treatment. 2 3 4 5

  21. Description of the action Audio Narration Step 4: T3: Addition of stain removal solution 1 2 3 Show like taking the tube from the mixer, after 30 minutes. Animate for the tube must contain colorless gel piece at the bottom and blue solution on top of it. User should click on the pipette to discard the blue solution in the tube and show like gel pieces inside the tube. Animate the stain removal technique like in above figure showing constitutes of Destaining solution acting on the gel piece. The destaining solution acts on gel pieces making it to swell and destain the gel pieces by reducing the interactions between protein and dye. 4 5

  22. Description of the action Audio Narration Step 5: T4: Solution Treatment 1 Animate like the user taking the tube in hand and showing the transparent gel pieces in inside it as shown in slide. Animate like the user taking the pipette and setting the value to 50ul. The user should click on the “dehydration solution” and open the lid to pipette out 50ul of the solution and adding it to the tube with gel pieces and keeping it stand for 5 minutes. After 5 minutes show like removing the liquid part and discarding, show whit pieces at the bottom, 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. Repeat the steps again. Show the clock running as per time specified The treatment must be repeated twice to ensure complete removal of water from the gel. 2 3 4 5

  23. Step 6: T4: Solution Treatment 1 2 60 C 3 Dry Bath 4 5

  24. Description of the action Audio Narration Step 6: T4: Solution Treatment 1 Animate like the user taking the tube in hand and showing the transparent gel pieces in 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 gel pieces and keeping it in a dry bath at 60’C for 1hr After 60 minutes show like removing the liquid part and discarding, show white pieces at the bottom, 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 to keep it for 60 minutes at 60’C, then remove the supernatant to add 50ul of 50mM ammonium bicarbonate to the tube and keep for 2minutes later remove the supernatant. Each time the solution is added to the gel pieces after the treatment the solution or supernatant must be discarded without disturbing the gel pieces. 2 3 4 5

  25. Description of the action Audio Narration Step 7: T4: Solution Treatment 1 Animate like the user taking the tube in hand and showing the transparent gel pieces in inside it as shown in slide 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 gel pieces and keeping it in room temperature in dark (animate accordingly) for 20 minutes After 20 minutes show like removing the liquid part and discarding, show white pieces at the bottom, now the user should click on the bottle labeled as “25mM ABC” and add 50ul (set) to it and show like leaving for 2minutes and show like the user removing the water layer after 2minutes. 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

  26. Description of the action Audio Narration Step 8: T4: Solution Treatment 1 Animate like the user taking the tube in hand and showing the transparent gel pieces in inside it as shown in slide. Animate like the user taking the pipette and setting the value to 50ul. The user should click on the “dehydration solution” and open the lid to pipette out 50ul of the solution and adding it to the tube with gel pieces and keeping it stand for 5 minutes. After 5 minutes show like removing the liquid part and discarding, show white pieces at the bottom, now the user should click on the bottle labeled as “25mM ABC” and add 50ul (set pipette) to it and show like leaving for 2 minutes. Repeat the steps again. Show the clock running as per time specified Add 50ul of dehydration solution and keep for 5 minutes , then remove the supernatant and add 50ul of 25mM ammonium bicarbonate to the tube and keep for 2 minutes, repeat the step twice to ensure complete removal of water from the gel 2 3 4 5

  27. Description of the action Audio Narration Step 9: ‏ 1 T4: Solution Treatment 2 3 Animate like the user taking the liquid part from the tube using the pipette when the user clicks on it and show like drying the pieces at the bottom in a “speed vac instrument as shown in figure. User should set the temperature (30’C), Time (15min) by clicking on the setting in the instrument. Please redraw the figure Concentrate the protein in speed vac instrument by drying it, removing any traces of the solution. 4 5

  28. Description of the action Audio Narration ‏ Step 10: T5: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 -80’C 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 user keeping the tube on ice bath. Add 1ml of 25mM ammonium bicarbonate solution to the 20ug of trypsin vial, which must be prepared fresh before the experiments. 2 3 4 5

  29. Description of the action Audio Narration Step 11: T5:Trypsin digestion 1 Show a tube with the pieces (transparent) and the 25mM ABC bottle and trypsin tube. The user must take the pipette ,click to set the value to 20ul and open the trypsin tube, take the amount by clicking on the pipette and show like adding to the tube with gel pieces and click on the user hand and instruct to keep in ice Show a clock running for 30 minutes The 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 gel pieces 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 gel pieces containing protein spots. Trypsin cleave the larger protein peptides into smaller peptides. For better trypsin digestion of the peptides a overnight treatment step is carried out. 2 3 4 5

  30. Description of the action Audio Narration Step 12: 1 T6:Extraction Show the bottle labeled as acetonitrile and formic acid. The user must take the pipette ,click to set the value to 500ul and open the acetonitrile bottle, take the amount by clicking on the pipette and show like adding to the tube labeled as “extraction solution “ as in slide:7. Again user must take the pipette, click to set the value to 2ul and open the formic acid bottle , take the amount by clicking on the pipette and show like adding to the tube “extraction solution “ and show like mixing as in slide:17. Prepare extraction solution consisting of 50% acetonitrile and 0.2% formic acid. The recipe is standard for carrying out the In-gel digestion experiment. 2 3 4 5

  31. Description of the action Audio Narration Step 13: 1 T6:Extraction Show the extraction solution tube after 16 hours animate like the user opening the incubator and taking out the tube. The user must take the pipette ,click to set the value to 100ul and open the extraction solution tube , take the amount by clicking on the pipette and show like adding to the tube with the gel pieces . Show like vortexing as shown in next slide Show like the user removing the liquid part (top layer) using the pipette and transferring to the new tube labeled as “trypsin digested sample”. Show like repeating the step for 2 more times. Show like performing Speed vac as in slide: 27 Extract the protein from the gel using extraction solution three times to ensure complete removal of the protein and perform speed vac to concentrate the protein. 2 3 4 5

  32. Step 14: T6:Extraction 1 2 3 4 Description of the action Audio Narration The user should click on the hand and place the tube and click start so that the tube will be vortexed. Show a clock running for 10 minutes. kindly redraw the figures Vortex the gel pieces with the extraction solution for 10 minutes. 5

  33. Step 15: T7: Zip-Tip 1 2 Column Audio Narration Description of the action 3 Show the box labeled as “Zip-Tip” as shown in figure. Instruct the user to click on the hand so that he takes a pipette and open the “zip-tip” box and take the tip as shown in next figure . The tip should be white in color at the end as shown in figure. Zoom in to show the column in the tip The column contains c18/c4 media for the peptide enrichment for MS analysis. 4 5

  34. Step 16: T7: Zip-Tip 1 2 Audio Narration Description of the action 3 Show a tube labeled as “acetonitrile” and the user should click on it to open and the user should set the pipette to 5ul and pipette the acetonitrile from the tube and discarding the pipette solution in the discard. Repeat the same step for 3 times and now show a tube labeled as “0.1% TFA” and the user should follow same step as mentioned above Pipetting should happen when the user clicks on the pipette Activate the zip tip column by using acetonitrile followed by 0.1% trifluoroacetic acid. 4 5

  35. Step 17: T7: Zip-Tip 1 2 Audio Narration Description of the action 3 Aspirate the zip-tip with the sample. Now show the tube labeled as sample and the user should set the pipette to 5ul when the user clicks on it , and show like pipette out the sample as and when the user clicks on the pipette 4 5

  36. Step 18: T7: Zip-Tip 1 2 Audio Narration Description of the action 3 Show a tube labeled as 0.1% TFA and the user should click on it to open and the user should set the pipette to 5ul and pipette the TFA from the tube and discarding the pipette solution in the discard. Repeat the same step for 3 times. Pipetting should happen when user clicks on the pipette. Wash the zip tip column using 0.1% trifluoroacetic acid to remove the storage material used to maintain the zip tip composition. 4 5

  37. Step 19: 1 T7: Zip-Tip 2 Audio Narration Description of the action 3 Show a tube labeled as 0.1% TFA and 50% ACN and the user should click on it to open and the user should set the pipette to 5ul and pipette the TFA/ACN from the tube and discarding the pipette solution in the tube labeled as “sample for MS". Repeat the same step for 3 times Pipetting should happen when the user clicks on the pipette Elute out the bound peptide using “0.1%Trifluoroacetic acid in 50% acetonitrile. 4 5

  38. Step 20: T8: Sample storage 1 2 Audio Narration Description of the action 3 Animate like the user taking the tube and placing it in box and opening a -20 C freezer to keep the box inside. Store the sample at -20’C until further analysis. Please follow the future viewing IDD for more information. 4 5

  39. Button 01 Button 02 Button 03 Slide 7-18 Slide 19-21 Slide 21-27 Slide 27-29 Slide 30-32 Slide 5-6 Slide 33-37 Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07 Name of the section/stage Animation area Interactivity area Slide 20-21 Show the user removing the acetonitrile within 15 minutes and the gel pieces look blue Instruction: Instruct the user to keep the gel pieces in acetonitrile for atleast 30 minutes Instructions/ Working area Credits

  40. Button 01 Button 02 Button 03 Slide 38-39 Tab 08 Tab 09 Tab 10 Name of the section/stage Animation area Interactivity area Instructions/ Working area Credits

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

  42. Questionnaire: APPENDIX 1 Question 4: Zip tip column contains a)C19 media b)C18 Media c)C5 Media d)C1 Media 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

  43. 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.

  44. APPENDIX 3 Summary In gel digestion involves the destaining of the spot to remove the stain , 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 extraction of the protein from the gel and concentrating the proteins.

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