Transcript 1 (a)

Quantitative proteomics SILAC
a
The identification and quantitation of complex protein
mixtures have been facilitated 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.
Harini Chandra
Master Layout (Part 1)
1
This animation consists of 2 parts:
Part 1: SILAC
Part 2: Application of SILAC
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Light medium
3
Harvest &
combine cells
Cell lysis &
proteolysis
Quantification
by MS
Intensity
Heavy
Light
m/z
Peptide
fragments
Mass spectrum
4
Heavy medium
5
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.
1
2
Definitions of the components:
Part 1- SILAC
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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4
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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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2
Part 1, Step 1:
Light medium
COO-
3
Normal/light
leucine
+
H3N
CH3
4
Action
5
Heavy medium
Cell culture
As
shown in
the
animatio
n.
C
COOH
+
D3N
C
CH2
CD2
CH
CD
CH3
Description of the action
First show the two flasks with the
colored solutions in them. The zoomed
in inset must be then appear which must
show the two structures as depicted.
The small circles must then appear in
the colored solution along with the
suitable label as shown in animation.
CD3
D
Deuterated/heavy
leucine
CD3
Audio Narration
SILAC is a simple method for in vivo incorporation
of a label into proteins for quantitative proteomic
purposes. Two groups of cells are cultured in
media that are identical in all respects except that
one contains a heavy, isotopic analog of an
essential amino acid while the other contains the
normal light amino acid.
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Part 1, Step 2:
Cell growth & replication
2
3
L
L
L
L
Proteins with
light leucine
4
Action
5
L
As
shown in
the
animatio
n.
Description of the action
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.
L
L
L
L
L
Proteins with
heavy leucine
Audio Narration
The essential amino acids which are obtained from
the cell culture medium are incorporated into the
corresponding newly synthesized proteins during
cell growth and replication. Medium containing the
heavy amino acids will give rise to heavy, isotopic
proteins.
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Part 1, Step 3:
Harvest & combine cells
Cultures mixed
2
3
10 min
2500 rpm
20oC
4
Action
5
As
shown in
the
animatio
n.
Description of the action
Show the liquids in the two flasks being poured
into the tube. This tube must then be placed in
the grey instrument which must be switched on
by pressing the green switch. When it is
pressed, the red text must appear. After this,
the tube shown on right bottom must appear
out of the instrument as shown.
Audio Narration
After a number of cell divisions, all instances of the
particular amino acid will be replaced by its
isotopic analog. The grown cells are then
combined together and harvested. Centrifugation
of the mixture will result in the pelleting of cells
which can then be used for further analysis.
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Part 1, Step 4:
Cell lysis & proteolytic digestion
Intact cells
2
Lysed cells
3
Lysis buffer
Trypsin
Peptide fragments
Light
4
Action
5
As
shown in
the
animatio
n.
Description of the action
Show the tube on left with only the colored circles at the bottom.
This must be zoomed into and the inset on the right must
appear. Next, the dark blue solution must appear as shown with
the label ‘lysis buffer’. When this happens, the outside coating of
the ‘intact cells’ must be broken down as shown in middle panel.
Next, the light blue solution must be added with label ‘trypsin’.
When this happens, the orange and red curved lines must be
fragmented into small pieces as shown.
Heavy
Audio Narration
The grown cells are then lysed using a
suitable lysis buffer and the proteins
degraded using a proteolytic enzyme
like trypsin. This results in a mixture of
light and heavy peptide fragments
which can be quantified suitably by
MS.
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Part 1, Step 5:
SDS-PAGE & in-gel digestion
SDS-PAGE
Separated peptide bands
2
Peptide fragments
3
Tubes with
buffer
Peptide fragments with
reduced complexity
4
Action
5
As
shown in
the
animatio
n.
Description of the action
Show the tube on the left with label. The small
fragments must then move on to the grey rectangle
where they must fade away and the blue figure with
dark bands on top must then appear. The dotted lines
must then appear as shown and a knife or scissors
(not shown here) must cut out the pieces. Each of the
fragment pieces must then enter the tubes below and
a red box must appear around the tube shown.
Audio Narration
The complex mixture of peptide fragments is
further separated by SDS-PAGE to simplify
the analysis. Each band of the gel is cut out
and re-dissolved in a suitable buffer solution.
These simplified peptide fragments are then
used for further analysis.
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Part 1, Step 6:
LC-MS/MS analysis
Peptide fragment
Column inlet
from pump
HPLC Pump
2
Injector
Mobile phase
3
Sample
LC Column
Sample injector
elution
Sample vials
4
Action
5
Pump Column
As
shown in
the
animatio
n.
Description of the action
Show the tube on top followed by the arrow. Then show the setup
below with all its labels. The second and third boxes must be
zoomed into to show the figures on the right. The ‘injector’ must
enter the sample bottle with its plunger down. It must remain in this
bottle for a couple of seconds and the plunger must be shown to
move up. This must then move and be injected into the column.
Liquid must be shown to flow through the tube connecting the ‘pump’
and ‘column’. Once the liquid flows, the colour in the column must
change and the liquid must be shown to pass through the tubing at
the outlet.
Column
Column outlet to
detector
Audio Narration
Further purification is carried out by liquid
chromatography wherein the sample is passed
through a column containing a packed
stationary phase matrix that selectively adsorbs
only certain analyte molecules. Reverse phase
and strong cation exchange chromatography
are the most commonly used. The eluted
fractions are further characterized by MS.
LC-MS/MS analysis
Heavy
Intensity
Light
Intensity
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Part 1, Step 7:
2
Heavy
Ratio of peak intensities is
indicative of ratio of protein
abundance.
Light
m/z
Detector
m/z
Peptide spectrum
3
m2
ESI
m3
Quadrupole
(scanning
mode)
4
Action
5
m4
m1
As
shown in
the
animatio
n.
Collision cell
TOF tube
Description of the action
First show all the components of the instrument – the syringe, four rods,
cube, blue rectangle, gray square with the dotted lines & the detector.
Next show appearance of the coloured circles. Only the red one must
move through the rods and after entering the rectangular box, it must be
fragmented to give smaller circles. These must migrate through the blue
tube and get reflected to reach the ‘detector’. The smallest circles must
move the fastest while the largest must move slowest. Once it reaches
the detector, the computer screen must appear with the figure shown.
This must be zoomed into and the figure on top left must be shown.
Reflector
Audio Narration
The purified peptide fragments are then
analyzed by MS/MS. Peptides containing
the heavy amino acid show higher m/z than
the corresponding light peptide fragments.
The pairs of identical peptides can be
differentiated due to the mass difference
and the ratio of peak intensities can be
correlated to the corresponding protein
abundance.
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Part 1, Step 8
MASCOT LC-MS/MS
data analysis
Search title
Sample protein
Enzyme Trypsin
Trypsin
Quantitation SILAC
Chymotrypsin
iTRAQ 4plex
Peptidase
Taxonomy Bacterial
SILAC
Mammalia
ICAT D8
Carboxymethyl (C)
Fixed
Bacterial
modifications
Plant
Database(s) SwissProt
NCBInr
MSDB
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Variable
modification
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Oxidation (M)
Peptide tol.
1.2
Data file
Data format
Instrument
4
Action Description of the
As shown
in
animaion.
Da
# C13
MS/MS tol. 0.2
Monoisotopic
Peptide charge
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Email [email protected]
Your name Proteomics
Da
Average
Choose file
ESI-Q-TOF
Precursor
Start search…
MALDI-TOF
ESI-Q-TOF
action
Audio Narration
MALDI-TOF-TOF
The MS/MS data analysis shareware has some extra inputs such as
First show the computer with the
screen having a form on the inside.
This must be zoomed into and the
form above must be displayed. Each of
the fields must be filled in as shown
with some requiring selection using the
white mouse pointer as depicted.
Quantitation, MS/MS tolerance, peptide charge, instrument etc. in addition to
the fields for PMF. They require inputs from the user regarding the
experimental parameters used such as enzyme cleavage, protein name,
modifications etc. and the desired search criteria like taxonomy, peptide
tolerance etc. Commonly used protein databases against which the MS
information is processed to retrieve sequence data include NCBI, MSDB and
SwissProt. The data file generated from MS is uploaded and the search
carried out.
Master Layout (Part 2)
1
This animation consists of 2 parts:
Part 1: SILAC
Part 2: Application of SILAC
2
SILAC
3
Heavy
Intensity
Haploid yeast cells
Light medium –
Normal L-lysine
Light
m/z
Peptide spectrum
4
Heavy medium –
L-lysine
13C /15N
6
2
Diploid yeast cells
5
de Godoy, L. M. et al., Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.
Nature 2008, 455 (7217): 1251-4.
1
2
Definitions of the components:
Part 2- Application of SILAC
1. Haploid yeast cells: The haploid number (n) is the number of chromosomes in a gamete. A
yeast having only ‘n’ chromosomes is said to be a haploid cell.
2. Diploid yeast cells: Yeast cells having two homologous copies of each chromosome (2n)
are said to be diploid cells.
3. Peptide spectrum: Once SILAC has been carried out on the haploid and diploid yeast cells
using light and heavy media, the peptide spectrum is generated following LC-MS/MS analysis.
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1
2
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Part 2, Step 1:
SILAC allows for labeling and
monitoring of dynamically changing
proteomes
Applications of
SILAC of sub-cellular organelles
which are involved in several
Signaling pathways
involving
activities
during apoptosis in cells.
kinases are employed in cell growth
and differentiation which play a
major role in cancer development
and progression. These pathways
and effects of inhibitors on them
have been
studied
using
SILAC.
Cellular
functions
are
mediated
by
several protein complexes that
interact with one other. SILAC has
be been applied for quantitative
determination of such complexes
and their interacting protein partners.
One of the more recent applications
of SILAC include the identification of
protease substrates using
differentially labeled bacterial cell
cultures.
SILAC provides an in vivo strategy
to label and monitor quantitative
differences at protein level in
different conditions, which has been
successfully employed for differential
profiling & biomarker identification.
Temporal dynamics of cell signaling
pathways that transmit information
through various PTMs, most
commonly reversible
phosphorylation,
Differential
expression of proteins &
Quantitative
proteomic
using
have been efficiently
studied bystudies
SILAC
identification
of
disease
biomarkers
have been carried out with
coupledSILAC
with MS.
Cell signaling dynamics
yeast, which serves as a model
organism for eukaryotic cells in
Studiesinto
on yeast
& its signaling
providing insights
biological
processes.pathways
Identification of methylation sites
Identification of protease substrates
Methylation, which is one of the
most common PTMs having
Studyvarious
of protein complexes &
biological roles, has been
interactions
successfully studied using
Analysis of signaling pathways &
isotopically labeled methionine
effects of pharmacological inhibitors
residues.
Subcellular proteomics
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Action
5
As
shown in
the
animatio
n.
Description of the action
First show the pie chart appearing followed
by highlighting of each of the segments and
appearance of the text in the call-outs as
shown. Finally, the purple segment must be
highlighted and must flash to indicate that
this is being emphasized.
http://silac.org/applications
Audio Narration
SILAC is a useful quantitative approach that has found
applications for several proteomic studies. < Text appearing in
each of the call-outs must then be narrated sequentially as that
segment of the pie chart is highlighted.>
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Part 2, Step 2:
Haploid yeast cells
2
Protein extraction,
digestion & LCMS?MS
Heavy
Intensity
Light medium –
Normal L-lysine
97.3% of proteome was
found to change less
than 50% in abundance
between haploid and
diploid cells!
3
Light
m/z
Peptide spectrum
4
Heavy medium – Diploid yeast cells
L-lysine
13C /15N
6
2
Action
5
As
shown in
the
animatio
n.
Description of the action
First show the flask on top with green
solution and the small pink cells. Then
show the flask below with the pink cells
which must be joined together as pairs.
Then show these being poured into the big
flask in the centre. Next the arrow must
appear with label and the graph on right
must appear with labels.
Audio Narration
The authors determined fold change of peptide pairs between
haploid and diploid yeast cells using SILAC. Labeled lysine
residues were used to grow the diploid yeast cells while haploid
cells were grown in normal lysine medium. The cultures were
mixed, proteins extracted and analyzed by LC-MS/MS. Protein
ratios between haploid and diploid cells were determined with
high accuracy. Comparison revealed that 97.3% of the proteome
changes less than 50% in abundance.
1
Interactivity option 1:Step No: 1 (a)
Normal natural lysinecontaining diet
Unlabelled mouse
2
13C
6
Hemoglobin
SILAC ratio
SILAC ratio
Human serum albumin
1
2
Action
5
Labelled mouse
Average lysine-6 incorporation over 4 weeks
3
4
lysine containing
diet.
As
shown in
the
animatio
n.
3
4
Weeks
1
2
Description of the action
Show the cartoon mice on top feeding on
the purple green pellets respectively. The
mouse feeding on the purple pellet must
remain unchanged while the mouse feeding
on green pellet must slowly turn red in
colour. Next, the normal mouse picture must
be shown as on right followed by the two
graphs shown below.
3
4
Weeks
Audio Narration
Kruger et al. tracked the incorporation of lysine-6 into the mouse
proteome over 4 weeks by providing a C-13 containing lysine
diet. Their development, growth and behaviour were observed in
addition to sampling various blood proteins . The labeled mice
were found to develop normally. Average lysine-6 incorporation
over 4 weeks in human serum albumin and hemoglobin is
depicted in the graphs.
1
Interactivity option 1:Step No: 1 (b)
What inference can be drawn from the difference in lysine-6 incorporation
between human serum albumin and hemoglobin in the mouse proteome?
A) Mouse hemoglobin does not develop normally.
2
B) Rate of incorporation of lysine is not calculated properly.
C) The lysine incorporation in human serum albumin is defective.
3
4
D) The long 60-day half life of mouse erythrocytes leads to less labeling of
hemoglobin.
Interacativity Type
Choose the correct
answer.
5
Options
User must choose one
of the four options
shown above.
Boundary/limits
Results
User must first be shown the animation as
described in 1 (a) along with the narration given in
that step. Once that is complete, user must be
given this question and asked to choose the
correct answer. The correct answer is (D). If the
user answers correctly, ‘correct answer’ must be
displayed but if user chooses the wrong answer,
then ‘wrong answer’ must be displayed and the
correct answer must be highlighted.
Krüger, M. et al., SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell
Function. Cell 2008, 134 (2): 353-364.
1
Questionnaire
1. Which of the following type of amino acids are labeled during SILAC?
Answers: a) Essential b) Non-essential c) Neutral d) Non-polar
2
2. The m/z difference between light and heavy Arginine is
Answers: a) 2 Da b) 6 Da c) 8 Da d) 10 Da
3. Which cell lines can be used for SILAC analysis
Answers: a) HeLa, b) C127, c) HEK293, d) none, e) all
3
4. Reverse phase chromatography is based on which of the following interactions?
Answers: a) Ionic b) Covalent c) Hydrophobic d) Hydrogen bonding
5. The function of DTT during in-gel digestion of proteins is:
4
Answers: a) Oxidation of disulphide bonds b) Cleavage at N-terminal of amino acids c) Cleavage at Cterminal of amino acids d) Reduction of disulphide bonds
6. 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
5
c) incorporation of isotope label is 100%
d) incorporation of isotope label is 50%
Links for further reading
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.
•
Krüger, M. et al., SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an
Essential Factor for Red Blood Cell Function. Cell 2008, 134 (2): 353-364.
•
de Godoy, L. M. et al., Comprehensive mass-spectrometry-based proteome quantification
of haploid versus diploid yeast. Nature 2008, 455 (7217): 1251-4.
•
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