Transcript Introduction of SILAC and its applications

Introduction of SILAC and its
applications
• Stable Isotope Labeling of Amino acids in
Culture
• Develop and promoted by Matthias Mann
• Two papers:
– Lipid Rafts (PNAS)
– Focal Adhesion precursors (Cell under review)
What does a mass spectrometer do?
Precise identification of mass
Can trap a single ion species and fragment to get subfragments (sequence)
Reminder of ICAT technology
Theoretically (database), know what the
fragments should be (Trypsin digest)
Purify over monomeric avidin column
(This is a selection/simplification scheme)
Ideally, should see ratios between state1 and
state2 (they will have the same retention time).
Check to see if the mass is correct.
Expect to see a ratio between the peak sizes.
SCX profile
LC profile of a single fraction
Composite of SCX separation.
Overview of Biological and Chemical Isotope Labeling Strategis
Two complementary samples
Labeling
Analysis
Example:
Have large analysis space
SILAC vs. ICAT
• Culture system only
• De Novo Proteins (no
serum contamination)
• No optimization
• Simplifies MS/MS
• More complete
peptide coverage
• ALL protein samples
• Labels selected
moieties
• Need to optimize
labeling
• Large linker group.
• Reduces complexity
How can you distinguish between isotoped species?
“Light”
“Light/heavy” mix
“Heavy”
Time-course of deuterated isoleucine incorporation.
Also gives you an indication of the robustness of the assay.
Can compare the peak strength.
How do you actually get quantitation?
1) Once you’ve identified the complementary peptides,
2) Backtrack and integrate the Xtracted Ion Chromatogram
Two papers
1) Unbiased Quantitative Proteomics of Lipid
Rafts Reveals High Specificity for Signaling
Factors
Foster LJ, de Hoog CL, Mann M
PNAS, 2003
2) RNA and RNA-binding proteins participate in
early stages of cell spreading through Spreading
Initiation Centers, sites of nascent focal adhesion
formation.
De Hoog CL, Foster LJ, Nielsen, MM, Mann M
(Cell), pending
Experimental Strategy:
1) Grow cells with “heavy” or “light” until saturation (5 divisions)
2) Add cholesterol depleting drugs (3 conditions)
1) Methyl-beta cyclodextrin
2) Filipin
3) nystatin
Biochemistry outline
1)
2)
3)
4)
5)
Lyse cells by either TX-100 or pH11 buffer.
Determine protein concentration and mix samples.
Purify lipid rafts.
Trypsinize
MS/MS analysis
Example of 2 proteins:
Doublet recognition
Identity
Abundance ratios
Enriched
Non-enriched
Global analysis of raft protein data:
MCD is most efficient at extracting
cholesterol
Ratio between experimental/control population
Detergeant
extractions:
1) MCD
2) Filipin
3) Nystatin
4) Average pH
extration
See sharp Cutoffs
This is the AMAZING table:
Clear demarcation of
Raft proteins
Associated Proteins
Non-specific proteins
ratios close to 1 (mostly
high abundance cellular
proteins (biochemical
background))
Serum proteins (not shown)
Raft proteins (1TM or fatty acid mod)
Associated proteins (no TM, but associate)
Positives and Negatives all make sense!!
Conclusions (from this paper and others):
Rafts enrich >10-fold for signalling proteins.
Rafts bind not just tyrosine kinases but also serine/threonine
kinases and G proteins (more ubiquitous than thought)
Focal adhesion protocol overview.
Sample difference is attachment
Then IP with talin, vinculin, paxillin.
Isotoped peptide identification
Isotoped peptide characterization (talin)
Peptide quantification (.83)
Protein results summary:
304 proteins identified:
Only a small number of proteins were attachment dependent.
(from SILAC quantitation data).
There are many problems holes in their quantitation data.
I’m not up to giving them a hard time today.
Cell biology….
Studied RACK1
Come back to what I think SILAC/ICAT criteria is…
-RACK1/vinculin interaction is
greater in adherent cells (2.06).
-In spread cells, they don’t
colocalize.
However, when cells are
DETACHED (1hr), then allowed
to SPREAD, they begin to
colocalize(2µm). (stage 1)
Focal adhesion begin to appear
under the colocalized spots
(stage2)
The cell spreads and
colocalization disappears
(stage3)
Similar results for paxillin, talin,
and FAK
Actin forms a ring around this
initial complex (SIC or spreading
initiation complex).
This ringstructure is transient and
gone by stage 3.
RNA binding proteins were
found reproducibly in all 3
Ips.
The RNA-binding proteins
hnRNP K, FUS/TLS,
hnRNP E1, SmB, SmD, but
NOT snRNP U1 were
found in SICs.
SICs stain for nucleic acids.
This is Rnase I sensitive.
In fully spread cells, don’t
see splicesome components
(part C).
SILAC vs. ICAT
• Culture system only
• De Novo Proteins (no
serum contamination)
• No optimization
• Simplifies MS/MS
• More complete
peptide coverage
• ALL protein samples
• Labels selected
moieties
• Need to optimize
labeling
• Large linker group.
• Reduces complexity
Which method would you use?
-SILAC can only be used in tissue culture.
If you are doing this, it simplifies the question (vs. ICAT) GREATLY.
-Cell lines vs. tumors.
Are the phenotypes and profiles from a tumor reflected on a dish?
Can solve lack of antibody problem temporarily…
-3-dimensional growth. SILAC can eliminate background completely!
-I believe this is the major advantage of SILAC (even without quantitation
-Criteria before proteomics
Proteomics is trivial/non-trivial!!! Just a glorified Western.
Information overload.
QUICK, CLEAN secondary screen.
Otherwise, proteomics is useless.
Just come up with interesting, unverifiable data!!!
Matrigel is useful and simple/complex
(~10mg/ml protein)