In gel digestion - OSCAR Main Page

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Transcript In gel digestion - OSCAR Main Page

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
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

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

1
2
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
3
3. Relate the digestion of peptides using trypsin
4. Plan the protein extraction step for mass
spectrometry analysis using Zip tips
4
5
5. Interpret the results of the experiment
6. Assess the troubleshooting steps involved in the
experiments.
1
2
Master Layout
Sample Thawing (Slide: 5-6)
R
Reagent preparation (Slide: 7-18)
Addition of stain removal
solution (Slide: 19-21)
3
Solution Treatment
(Slide: 22-27)
Trypsin digestion (Slide: 28-29)
4
Extraction (Slide: 30-32)
Zip-Tip (Slide: 33-37)
5
Storage (Slide:38)
Display a image from each of these steps, with user click.
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2
3
4
5
Definitions and Keywords
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.
1
Step 1:
T1: Sample Thawing
2
3
4
5
Description of the action/
interactivity
Animator should instruct the user
to go through the IDD-26. Spot
picking prior to start this
experiment.
Audio Narration
(if any)
User must be aware of the
technique involved in spot picking
and storage of excised spot.
1
Step 2:
T1: Sample Thawing
Description of the action
2
3
4
5
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.
Audio Narration
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.
1
Step 3:
T2: Reagent preparation
2
3
Description of the action
4
5
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.
Audio Narration
Label the 15ml falcon tubes
as “staining solution,
dehydration solution,
reduction solution, alkylation
solution, extraction solution”
prior to reagent preparation.
1
Step 3:
T2: Reagent Preparation
2
3
Description of the
action
4
5
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”
Audio Narration
(if any)
Clean the surface of
the balance, Tare the
weight of the paper
before weighing.
Video file: Balancing
1
Step 3:
T2: Reagent Preparation
2
Beaker
Magnetic
bead
3
Description of the
action
4
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.
5
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
1
2
3
4
5
Step 3:
T2: Reagent Preparation
ABC
Water
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.
Audio Narration
Weigh 0.197 g of
ammonium
bicarbonate. Give a
brief spin to dissolve
powder completely
and later make the
volume to 100ml.
1
2
Step 3:
T2: Reagent Preparation
ABC
Water
3
4
5
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.
Audio Narration
Weigh 0.395 g of
ammonium
bicarbonate. Give a
brief spin to dissolve
powder completely
and later make the
volume to 100ml.
1
2
3
4
5
Step 3:
T2: 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 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.
Water
Audio Narration
Weigh 0.790 g of
ammonium bicarbonate.
Give a brief spin to
dissolve powder
completely and later
make the volume to
100ml.
1
2
3
4
5
Step 3:
T2: Reagent Preparation
DTT
Description of the action
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.
Water
Audio Narration
Prepare 10mM DTT
using 100mM ammonium
bicarbonate and mix
them well.
1
2
3
4
5
Step 3:
T2: Reagent Preparation
IAA
Description of the action
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.
Water
Audio Narration
Prepare 50mM
iodoacetamide using
100mM ammonium
bicarbonate and mix
them well.
1
Step 3:
T2: Reagent Preparation
Description of the action
2
3
4
5
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.
Audio Narration
Prepare 0.1% Trifluoroacetic acid
using Milli-Q water.
1
Step 3:
T2: Reagent Preparation
Description of the action
2
3
4
5
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”.
Audio Narration
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.
1
Step 3:
T2: Reagent Preparation
2
3
4
5
TUBE
(destaining
solution)
1
Step 3:
T2: Reagent Preparation
Description of the action
2
3
4
5
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”.
Audio Narration
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.
1
2
3
4
5
Step 4:
T3: Addition of stain removal solution
1
Step 4:
T3: Addition of stain removal solution
Description of the action
2
3
4
5
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.
Audio Narration
Add 200ul of destaining
solution and mix the
contents for 30 minutes for
better treatment.
1
Step 4:
T3: Addition of stain removal solution
2
3
Description of the action
4
5
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.
Audio Narration
The destaining solution acts
on gel pieces making it to
swell and destain the gel
pieces by reducing the
interactions between protein
and dye.
1
Step 5:
T4: Solution Treatment
Description of the action
2
3
4
5
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
Audio Narration
The treatment must be
repeated twice to ensure
complete removal of water
from the gel.
1
Step 6:
T4: Solution Treatment
2
3
4
5
Dry Bath
1
Step 6:
T4: Solution Treatment
Description of the action
2
3
4
5
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
Audio Narration
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.
1
Step 7:
T4: Solution Treatment
Description of the action
2
3
4
5
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
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.
1
Step 8:
T4: Solution Treatment
Description of the action
2
3
4
5
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
Audio Narration
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
1
Step 9:
T4: Solution Treatment
2
3
Description of the action
4
5
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
Audio Narration
Concentrate the protein in
speed vac instrument by
drying it, removing any
traces of the solution.
1
Step 10: T5:Trypsin digestion
Description of the action
2
3
4
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.
5
Audio Narration
Add 1ml of 25mM
ammonium bicarbonate
solution to the 20ug of
trypsin vial, which must
be prepared fresh before
the experiments.
1
Step 11: T5:Trypsin digestion
Description of the action
2
3
4
5
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
Audio Narration
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.
1
Step 12:
T6:Extraction
Description of the action
Show the bottle labeled as
acetonitrile and formic acid.
2
3
4
5
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.
Audio Narration
Prepare extraction solution
consisting of 50%
acetonitrile and 0.2%
formic acid. The recipe is
standard for carrying out
the In-gel digestion
experiment.
1
Step 13:
T6:Extraction
Description of the action
Show the extraction solution tube
2
3
4
5
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
Audio Narration
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.
Step 14:
1
T6:Extraction
2
3
4
5
Description of the action
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
Audio Narration
Vortex the gel pieces
with the extraction
solution for 10
minutes.
1
Step 15: T7: Zip-Tip
2
3
4
5
Description of the action
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
Column
Audio Narration
The column contains
c18/c4 media for the
peptide enrichment for
MS analysis.
1
Step 16: T7: Zip-Tip
2
3
4
Description of the action
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
5
Audio Narration
Activate the zip tip
column by using
acetonitrile followed by
0.1% trifluoroacetic
acid.
1
Step 17: T7: Zip-Tip
2
3
4
5
Description of the action
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
Audio Narration
Aspirate the ziptip with the
sample.
1
Step 18: T7: Zip-Tip
2
3
4
Description of the action
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.
5
Audio Narration
Wash the zip tip column
using 0.1%
trifluoroacetic acid to
remove the storage
material used to
maintain the zip tip
composition.
1
Step 19:
T7: Zip-Tip
2
3
4
Description of the action
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
5
Audio Narration
Elute out the
bound peptide
using
“0.1%Trifluoroacet
ic acid in 50%
acetonitrile.
1
Step 20: T8: Sample storage
2
3
4
5
Description of the action
Animate like the user taking the tube and
placing it in box and opening a -20 C
freezer to keep the box inside.
Audio Narration
Store the sample at 20’C until further
analysis. Please
follow the future
viewing IDD for more
information.
Slide 56
Tab 01
Slide 718
Tab 02
Slide
19-21
Tab 03
Slide
21-27
Tab 04
Slide
27-29
Slide
30-32
Tab 05
Slide
33-37
Tab 06
Tab 07
Name of the section/stage
Interactivity
area
Animation area
Slide 20-21
Show the user removing the acetonitrile within 15 minutes and the gel pieces
look blue
Button 01
Button 02
Instruction:
Instruct the user to keep the gel pieces in acetonitrile for atleast 30 minutes
Button 03
Instructions/ Working area
Credits
Slide
38-39
Tab 08
Tab 09
Tab 10
Name of the section/stage
Interactivity
area
Animation area
Button 01
Button 02
Button 03
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1:
What is the main constituent of reduction solution?
a) Dithiothreitol
b) Iodoacetamide
c) Ammonium Bicarbonate
d) Trypsin
Question 2:
Reaction that occurs between iodoacetamide and disulphide bond
a) Alkylation
b) Acetylation
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:
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
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.
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.