Download Stable Isotope Labeling by Amino acids in Cell culture (SILAC)

Methodology for Stable Isotope Labeling by Amino
acids in Cell culture (SILAC)
The quantitation and identification of complex protein within the mixtures have
been helped by mass spectrometric methods based on differential stable
isotope labelling. These tags, which can be recognized by MS, provides a
basis for quantification. Stable Isotope Labeling by Amino acids in Cell culture
(SILAC) incorporates specific labelled amino acids into proteins for differential
analysis.
 Related Los: Isobaric tag properties, Trypsin properties
> Prior Viewing – IDD-2. Extraction of plant protein, IDD-27. In gel digestion, IDD-28.
In solution digestion
Future Viewing – IDD-30. Matrix Instrumentation, IDD-31. MALDI-TOF data
analysis, IDD-39. LC-MSMS data analysis
>
 Course Name: Stable Isotope Labeling by Amino acids in Cell culture (SILAC)
 Level(UG/PG): PG
 Author(s) : Dinesh Raghu, Vinayak Pachapur
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contents inDr.
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Sanjeeva
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Definitions and Keywords
1. Stable Isotope Labeling by Amino acids in Cell culture (SILAC): SILAC is a simple and
convenient method for in vivo incorporation of a suitable label into proteins for quantitative MSbased proteomics. Two groups of cells are grown in cultures that are identical in all respects
except that one contains a light medium with regular, unmodified essential amino acid while
the other contains a heavy medium, in which a heavy isotopic form of the amino acid is
present.
2. Light medium: Cell culture medium containing the regular, unmodified forms of all the
amino acids.
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3. Heavy medium: Cell culture medium in which labelled analogs of certain essential amino
acids are supplied to cells (for eg. Leucine-d3, arginine-C13). These amino acids get
incorporated into the proteins after a number of cell divisions and can be used to determine
the relative protein abundance by measuring MS signal intensities between corresponding
light and heavy peptides.
4. Cell lysis & proteolysis: The cells that have been grown in light or heavy medium are
lysed using a suitable lysis buffer and the proteins then digested using enzyme such as
trypsin. Peptide fragments of suitable length are generated for analysis by MS.
5. Quantification by MS: The peptide fragments obtained after proteolytic digestion are then
subjected to analysis by suitable mass spectrometry techniques. The intensity of MS signals
obtained for light and heavy peptides is directly related to the relative protein abundance.
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Learning objectives
After interacting with this learning object, the learner will
be able to:
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Perform culturing of cells using the heavy and light
amino acids
2.
Carry out Protein extraction from the cultured cells
3.
Define inoculation of growth in fresh media
4.
Carry out SDS-PAGE gel run
5.
Perform samples through chromatography
6.
Analysis of data and interpretation of the results
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Master Layout
Slide
5-11
Sample processing
Slide
12-16
Sample labelling
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Slide
17
SDS-PAGE
Slide
18-20
Liquid chromatography
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5
Slide
21-23
Data analysis and interpretation
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Step 1:
T1:Media preparation
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Description of the action
Audio Narration
(if any)
Text to be
displayed
Show a measuring balance, with display, ON,
When measuring
OFF and TARE/0 buttons on it. let user ON it,
display reading as 0.000g, let user picks up the with paper, the
weight of the
paper from the rack, makes 1/10 of folding on
paper need to be
the sides and places it on the balance. Now
the display reading changes to 0.003g. Instruct tarred from actual
reading.
user to TARE the reading. And animate to click
the tare button. Once user clicks it, reading
must show ”0”
Measuring
balance
Video file: Balancing
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Step 1:
T1:Media preparation
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Description of the action/ interactivity
Show a person sitting in front of laminar hood,
zoom the laminar working bench having , tissue
roll, ethanol bottle, burner, beaker with tooth picks.
Instruct user to clean the bench. Allow user to pick
tissue, wet it with ethanol and clean the whole
working bench with user control the user should
click on the hand so that the cleaning must
happen. Click for ON button for “light” and “blower”
to start the laminar hood to perform the
experiment. Please redraw the figures
Audio Narration
(if any)
Clean the laminar air
flow thoroughly with
ethanol
Video file: Laminar air flow
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Step 1:
T1:Media preparation
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Description of the action/ interactivity
Instruct user to prepare Amino acid stocks. Let user take out
PBS buffer, light, medium and heavy label amino acids from
the rack of laminar hood. Let user take PBS bottle, take out
5ml in fresh 15ml falcon tube. Measurements to be done
using measuring cylinder with user interactions. let user weigh
150mg of each light amino acid separately and add to the
falcon. Similarly let user do it for medium, heavy amino acid
also. say user want to use 1 light, 1 medium and 1 heavy
amino acid. So user need to have 3 falcon tubes, with 5ml of
PBS, with 150mg of each heavy, medium and light amino
acids label the tube accordingly at last. For each falcon
volume, set 0.5ml pipette and prepare aliquots in fresh tubes,
let user label and store at -80’C freezer. Animate preparation
in laminar hood.
Audio Narration
The heavy and light
label amino acid are
available from the
market, depending
upon user experiment
choices can be made.
The amino need to be
prepared in PBS and
made aliquots can be
stored at -80’c for
2months.
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Step 1:
T1:Media preparation
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Description of the action/ interactivity
Instruct user to prepare “SILAC medium”. Let user take out
culture medium(amino acid depleted), light/medium/heavy
amino acids aliquots, fetal calf serum and antibiotics on the
rack of laminar hood. Place them over the bench of laminar
hood. To the 500ml culture medium in conical flask, let user
measure and add 50ml of calf serum, with 5ml and 0.5ml set
pipette let user add antibiotics. Measurements to be done
using measuring cylinder with user interactions. Let user
prepare three sets of SILAC medium, in one let user set the
pipette to 0.5ml and add light amino acid solution, in other let
user set the pipette to 0.5ml and add medium light amino acid
and in other let user set the pipette to 0.5ml to add heavy
amino acid. And label the culture accordingly. Animate the
preparation in laminar hood.
Audio Narration
Depending upon the
type of cell growth, the
culture medium can be
defined. Now SILAC
medium for both heavy,
medium and light amino
acid isotopes are ready
for the inoculation.
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Step 1:
T1:Media preparation
filter membrane
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Description of the action/ interactivity
Instruct user to pass the “SILAC medium” through the
filter. Please re-draw the filter unit, let user take out 0.22um filter membrane and place it in the unit. Place the clip,
now let user add the media prepared from previous slide
on top of filter unit. Animate, the liquid solution getting
passed through the filter aid, and dropping into the
collection unit drop by drop.
Audio Narration
The media prepared is
passed through the
filter aid to remove any
contamination. The
collected solution can
be taken out and used
for inoculation. In some
case user can carry out
autoclave also.
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Step 1:
T1:Media preparation
autoclave
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Description of the action/ interactivity
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Instruct user to carry out autoclave of media. Let user
plug in a cotton roll on top to seal the opening of
conical flask. Now cover the opening with paper, place
a rubber to fix the paper and the cotton roll tightly. Let
user open the lid of the autoclave, let user check the
water level, let user add the water to makeup the
volume, now let user place the conical flask and close
the lid of the autoclave. Now animate the display unit
for time and temperature for user to set the
parameters. Let user set the time for 15min and
temperature for 121’C and press enter. Display a
gradual increase of temperature from 37’C to 121’C.
once temperature reaches 121’C run the time for
15min to carry out the autoclave.
Audio Narration
The media prepared
need to autoclaved
to be free of
contaminants.
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Step 1:
T1:Media preparation
autoclave
Description of the action/ interactivity
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Instruct user to take out conical flask from autoclave.
Let user loosen the pressure valve at top to release
excess steam inside the autoclave. Open the lid of
autoclave take out the flasks and keep it on laminar
hood for the inoculation.
Audio Narration
Once autoclaved, let
the media, cool
downs to room
temperature to be
used for inoculation.
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Step 2:
T2:Inoculation and protein extraction
Bacterial
culture
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SILAC
medium
Description of the action/ interactivity
Take user through the IDD:1 Extraction of Bacterial protein
from slides 6 to 7. animate inoculation of cell type in three
SILAC medium, one for heavy, other for medium and last
for light label amino acid. Redraw the figure for SILAC
medium in conical flask. Please do the animation
accordingly.
Audio Narration
(if any)
Place the inoculated
flasks in the shakerincubator at 37’C for
24hrs.
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Step 1:
T1:Media preparation
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Description of the action/ interactivity
First show the three flasks with the colored
solutions in them. The zoomed in inset
must be then appear which must show the
three structures as depicted. The small
circles must then appear in the colored
solution along with the suitable label as
shown in animation.
Audio Narration
SILAC is a simple method for in vivo
incorporation of a labelled amino acids
into the proteins for quantitative
proteomic analysis. Three groups of
cells are cultured in media that are
identical in all respects except that
one contains a heavy, isotopic analogy
of an essential amino acid while the
other contains the normal light amino
acid and the other contains medium
amino acid.
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Step 1:
T1:Media preparation
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Description of the action/ interactivity
Show both the flasks with more number of
colored circles in each. One of the circles must
be zoomed into and the figures shown in inset
below must appear. Animate the leucine amino
acid/circles taken up cell and getting incorporated
into the protein structure. During the time show
only fewer circles present in each flask.
Audio Narration
The amino acids requirement
from the cell culture medium
are incorporated into the
cells during the synthesis of
proteins during cell growth
and replication. For example
medium containing the
heavy amino acids will give
rise to heavy isotopic
proteins.
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Step 2:
T2:Inoculation and protein extraction
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Description of the action/ interactivity
After 24hrs, instruct user to take out 1ml from
each flask for the fresh inoculation. Let user
take the conical flasks to laminar hood, take
out 1ml from each flask and inoculate the
growth into fresh SILAC medium, repeat the
step in slide:12 and keep the incubation period
for 1day. Repeat this step for 3more days
until, user gets better separation in MS
analysis.
Audio Narration
(if any)
After each day of growth,
sample must be taken for
inoculation into fresh media.
Meanwhile the growth is going
on, protein analysis must be
carried out simultaneously. The
inoculation need to carried out
until user get the best MS result
for incorporation of heavy amino
acids in comparison with light
ones.
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Step 2:
T2:Inoculation and protein extraction
Day:1
5ml
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Description of the action/ interactivity
After the inoculation, instruct user to take out 5ml from each
flask for the analysis. Let user take the conical flasks to
laminar hood, take out 5ml from each flask in measuring
cylinder, mix and pool in fresh falcon for protein extraction.
In case if the culture is grown in Petri plate, scrap the
growth to take out the sample into the falcon and carry out
protein extraction. For the protein extraction, take user
through the IDD:1 Extraction of Bacterial proteins from
slides: 8 to 27.
Audio Narration
(if any)
Once the cells are
harvested, grown,
combine into one, do
the cell purification, cell
lysis, follow the protein
extraction and
solublizie in
rehydration buffer to
carry out SDS-PAGE
or In-solution digestion.
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Step 3:
T3:Protein separation
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Description of the action/ interactivity
After getting sufficient amount of protein
concentration, Instruct user to carry out
SDS-PAGE run for the samples from day 1.
Take user through the IDD:17SDS-PAGE
for protein separation. Animate the SDSPAGE gel with blue bands at different
regions along the gel. Let user do the
manual picking please refer IDD:26 Spot
picking and carry out trypsin digestion like
in IDD:27 In-gel digestion.
Audio Narration
(if any)
SDS-PAGE sample loading buffer:
0.3 M Tris–HCl, pH 6.8,
10% SDS, 12.5% vol/vol 2mercaptoethanol, 50% glycerol,
0.016% bromophenol blue
Depending upon user set up, one
can carry to resolve protein sample
through SDS-PAGE followed by ingel trypsin digestion to carry out MS
or directly can do In-solution trypsin
digestion followed by MS analysis.
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Step 4:
T4:Liquid chromatography
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Description of the action/ interactivity
Display the figure above of HPLC unit, for
more information user can follow IDD: 41
Liquid chromatography. Animate user
injecting the sample into the instrument,
now along with mobile sample must move
into the column, in the column show the
separation of peptides from one another,
like one peptide eluting out faster and
other slower, the peptide must be
collected into the sample vials.
Audio Narration
(if any)
Instrument setup: system equipped with a
capillary pump, micro-vacuum degasser,
microwell-plate autosampler. The sample
peptides are eluted out or separated out
from one other depending on the binding
properties of peptide to the stationery
phase. The retention and elution properties
of the peptide help in the separation by
controlling the flow rate of mobile phase with
the help of pump. The separated peptide
are collected into each sample vials
separately. nowthe samples are ready for
the MALDI-MS analysis.
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Step 4:
T4:Liquid chromatography
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3
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5
Description of the action/ interactivity
For LC-MS setup and parameters details:
Mobile phase A – 0.4% acetic acid +
0.005% heptafluorobutyric (vol/vol) in
water. Mobile Phase B- 90% acetonitrile,
0.4% acetic acid and 0.005%
heptafluorobutyric (vol/vol) in water.
Column details: inner diameter 75um,
length 10cm, flow rate 300nl/min and
back pressure 50-150bar. For complex
sample, long gradient: 10% solvent B to
45% solvent B for 90min. For low
complexity, a gradient: 10% solvent B
increasing to 45% solvent B for 45min.
Audio Narration
(if any)
Based on LC system, the column dimension
and flow rates vary. Let user set appropriate
LC gradient depending upon the sample
complexity. Performance of the LC must be
checked in-between by running the
standards.
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Step 4:
T4:LC
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3
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5
Description of the action/ interactivity
For firing of sample spots and data acquisition
please go through IDD: MALDI instrumentation
and LC-MS/MS. For the image above animate,
firing of isotope bind to peptide being fired and
detected as identical peak in MS.
For data acquisition, let user perform the LC
system on peptide elution width. Let user
estimate the number of scans per eluting
peptide. Animate for three MS scans within the
duration of a peptide elution. Set the
parameters for data acquisition depending on
LC system used.
Audio Narration
(if any)
The incorporation data for each day
must be analysed. In the spectrum
obtained, the incorporation of
medium and heavy amino acid
molecule is must be more intense as
compared to light.
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2
3
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Step 5:
Light region
T5:Data analysis and interpretation
Heavy region
Description of the action/ interactivity
Audio Narration
(if any)
1
Step 5:
T5:Data analysis and interpretation
Description of the action/ interactivity
Audio Narration
(if any)
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3
Animate the figure from previous slide for each
day of MS analysis. Animate for day 1
spectrum showing light amino acid peaks with
intensity half the heavy amino acid peak. From
Day2 to Day6 show the decrease in the
intensity of the light amino peaks compared to
heavy ones.
For Protein identification, let user go through
IDD:31 MALDI-TOF Data analysis
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So in the initial day of MS analysis,
light amino acids show some
incorporation along with heavy ones.
Later with progress of experiment the
cells tend to take up the heavy ones,
its incorporation as compared to light
ones is more. This sort of analysis
can be used to compare control and
disease samples, for protein
expression.
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Step 5:
T5:Data analysis and interpretation
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3
4
5
Description of the action/ interactivity
For data analysis and interpretation please go
through IDD:31- MALDI TOF data analysis.
Search the processed MALDI- MS data against
the relevant protein database by taking all the
required modifications into consideration.
Audio Narration
(if any)
After collection of spectrum, protein
need to be identified with help of
database search. Once protein
identification is done, user can
correlate the proteins for the study.
Slide
1-4
Introduction
Slide
5-11
Tab 01
Slide
12-16
Tab 02
Slide
17
Tab 03
Slide
18-20
Tab 04
Slide
21-23
Tab 05
Tab 06
Name of the section/stage
Interactivity
area
Animation area
Ask user for potential problems during the experiment.
Instruction: Improper separation: if OFFGEL: increase the
number of fractions from 12-24, if 2D: lengthened
gradient profile, if LC: longer column with finer particle
size.
Button 01
Button 02
Button 03
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
1. The m/z difference between light and heavy Arginine is (with which isotopic
atom??)
Answers: a) 2 Da
b) 6 Da
c) 8 Da
d) 10 Da
2. Which cell lines can be used for SILAC analysis
Answers: a) HeLa,
b) C127,
c) HEK293,
d) none,
e) all
APPENDIX 1
Questionnaire:
3. Which of the following type of amino acids are labeled during SILAC?
Answers: a) Essential
b) Non-essential
c) Neutral
d) Non-polar
4. Which of the following statements concerning SILAC is incorrect?
Answers: a) no chemical difference between labeled and natural amino acid
isotopes
b) cells behave exactly like control cell population grown in presence of
normal amino acid
c) incorporation of isotope label is 100%
d) incorporation of isotope label is 50%
APPENDIX 2
Research papers:
•Ong, S. E. et al., Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple
and Accurate Approach to Expression Proteomics. Mol. Cell. Proteomics 2002, 1:376-386.
•Kerner, M. J. et al., Proteome-wide analysis of chaperonin-dependent protein folding in
Escherichia coli. Cell 2005, 122 (2): 209-20.
•Harsha, H. C., Molina, H. & Pandey, A. Quantitative proteomics using stable isotope labeling
with amino acids in cell culture. Nat. Protoc. 2008, 3: 505-516.
Websites:
•http://www.silac.org
APPENDIX 3
Summary
The quantitation based on SILAC proteomic approach depends on various
parameters including the selection of both light and heavy amino acids, the
choice of proteolytic enzyme trypsin protease, protein separation based on 1D,
In-solution digestion, normalization of proteins samples and depth of mass
spectrometric analysis.