Transcript In solution digestion

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
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. MALDITOF data analysis
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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

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Learning objectives
After interacting with this learning object, the learner
will be able to:
1. Define the destaining of the selected spots
2. Recognize to reduce and alkylate the proteins using
chemical reagents
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3. Relate the digestion of peptides using trypsin
4. Plan the protein extraction step the gel for mass
spectrometry analysis
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5. Interpret the results of the experiment
6. Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Reagent Preparation
(Slide: 5-12)
Sample processing (Slide: 13-16)
Reduction and alkylation
(Slide: 18-19)
Trypsin digestion (Slide: 20-21)
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Stopping the reaction (Slide: 22)
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Storage (Slide: 23)
Display a image from each of these steps, with user click.
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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.
2) Alkylation solution: It consists of iodoacetamide which prevents the
reformation of disulfide bond.
3) 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.
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Step 1:
T1: Reagents preparation
Water
Tris-HCl
Tris-Hcl
PH 8
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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
Audio Narration
The recipe is standard for
carrying out the In-solution
digestion experiment.
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Step 1:
T1: Reagents preparation
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Beaker
Magnetic
bead
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Description of the
action
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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
Audio Narration
(if any)
The magnetic stirrer helps
for the evenly distribution of
the solute into the solution.
Video file: Magnetic stirrer
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Step 1:
T1: Reagents preparation
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NaOH
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HCl
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.
Audio Narration
Prepare pH-8 Tris-HCl
solution, the exact pH is
required for better
results.
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Step 1:
T1: Reagent Preparation
ABC
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 (0.790g)
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
Water
Audio Narration
ABC is used to for reducing
the interactions between
protein and dye.
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Step 1:
T1: Reagent Preparation
DTT
Description of the action
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.
Water
Audio Narration
Prepare 100mM DTT using
100mM ammonium
bicarbonate (ABC) and mix
them well.
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Step 1:
T1: Reagent Preparation
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IAA
Description of the action
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
Water
Audio Narration
Prepare 100mM
Iodoacetamide (IAA) using
100mM ammonium
bicarbonate and mix them
well. Different reagent
preparation are used for
protein treatment.
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Step 1:
T1: Reagent Preparation
Description of the action
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Show a bottle labeled as “acetic acid”
and measuring cylinder.
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”
Audio Narration
Please make calculation for
0.5% acetic acid depending
upon the sample size as it is
used for the extraction step.
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Step 1:
T1: Reagent Preparation
Description of the action
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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.
Audio Narration
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.
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Step 2:
T2:Sample Processing
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Description of the action/ interactivity
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.
Audio Narration
(if any)
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.
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Step 3:
T2: Sample processing
Description of the action
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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.
Audio Narration
Add 100mM ammonium bicarbonate
and 5% acetonitrile to the sample. This
is to de-stain the sample solution as a
additional step.
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Step 3:
T2: Sample processing
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Show the color as
dark green than blue
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Description of the action
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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
Audio Narration
The pH of the sample should be 8
for better trypsin activity.
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Step 3:
T2: Sample processing
Description of the action
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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.
Audio Narration
If the pH is acidic , add tris –hcl
bring down to pH:8 to make it
“basic pH”
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Step 4:
T3: Reduction and alkylation
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Dry Bath
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Please re-draw the figure. Tube labeled as reducing agent…not dehydration solution
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Step 4:
T3: Reduction and alkylation
Description of the action
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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 510minutes.
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
Audio Narration
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
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Step 5:
T3: Reduction and alkylation
Description of the action
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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
Audio Narration
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.
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Step 6:
T4:Trypsin digestion
Description of the action
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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)”
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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
Audio Narration
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.
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Step 7:
T4:Trypsin digestion
Description of the action
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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.
Audio Narration
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.
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Step 8:
T5: Stopping the reaction
Description of the action
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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.
Audio Narration
Acidify the trypsin digest
mixture to cease the reaction
using 0.5% acetic acid.
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Step 9 :
T6:Sample storage
sample
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Description of the action
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
Audio Narration
Store the concentrated sample until
further usage.
Please follow the future viewing IDD
for more information
Slide 512
Tab 01
Slide 13
Tab 02
Slide
14-16
Tab 03
Slide
17-18
Tab 04
Slide 19
Tab 05
Slide
20,21
Slide
22-23
Tab 06
Tab 07
Name of the section/stage
Interactivity
area
Animation area
Animate a question like “ If the protein extract is in the buffer , trypsin digestion
can be done by
Button 01
Give options like “ In gel digestion and In solution digestion “
Button 02
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”
Button 03
If the user click on the In solution digestion “take him to the animation of in
solution digestion”
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1:
What is the main constituent of alkylation solution?
a) Dithiothreitol
b) Iodoacetamide
c) Ammonium Bicarbonate
d) Trypsin
Question 2:
Reaction that occurs between Dithiothreitol and disulphide bond
a) Alkylation
b) reduction
c) Iodine addition
d) Denaturation
Question 3:
Trypsin is a
a)
b)
c)
d)
Catalyst
Inhibitor
Protease enzyme
Gel constituent
APPENDIX 1
Questionnaire:
Question 4:
0.5% acetic acid is used for
a)Increase the pH of trypsin-sample mixture
b)Decrease the pH of trypsin–sample mixture/to stop or reduce the activity of trypsin
c)Neutralize the pH
d)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
APPENDIX 2
Links for further reading
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Reference websites:
http://iitb.vlab.co.in
Books:
Andrew j.Link and Joshua LaBaer. “Proteomics”
Cold spring harbor laboratory manual.
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 .