Project 8
Signal-dependent interactions in insulin action
Primary Investigator: Matthias Mann, Ph.D.
University of Southern Denmark
Specific Aims:
1. Measure proteome-wide changes in phosphorylation triggered by
insulin in white and brown adipocyte cell lines.
2. Compare changes of the insulin-induced tyrosine phosphoproteome in
normal brown adipocytes and in cells with specific knock out for key
insulin signaling factors.
3. Identify proteins interacting specifically with insulin-induced
phosphotyrosine moieties.
Summary:
Insulin binding to its receptor initiates a complex network of events, starting
with a tyrosine phosphorylation cascade that branches out to affect multiple
endpoints. It is likely that many steps of the insulin pathway are adversely
affected in various forms of type II diabetes. However, the polygenic nature of
the disease makes it difficult to understand its molecular level. While gene
expression methods are very well developed, they do not address many of the
changes that may occur in cell signaling. To study changes in protein
abundance, we are using a combination of nano-flow high performance liquid
chromatography, sensitive mass spectrometers (LTQ-FTMS), and the SILAC
method (Stable Isotope Labeling by Amino acids in Cell culture). These
techniques will allow us to define changes in the insulin-dependent tyrosine
phosphorylation on a proteomic scale.
Protocols
1. Culture of brown adipocytes (BAT) in SILAC Medium and Insulin
induction
2. Immunoprecipitation
3. In Gel digestion
4. Sample preparation after “in gel” digestion
5. HPLC system and mass spectrometer
6. More information about SILAC
1. Culture of brown adipocytes (BAT) in SILAC Medium and Insulin
induction
The brown adipocytes cell lines were kindly provided by Ronald Kahn. The
protocol for adipocyte differentiation is adopted from Ronald Kahn´s lab
(see also Project 1 / Brown fat cell isolation).
SILAC Media:
DMEM based on Cat #31885-023 (GIBCO) but
WITHOUT L-lysine, and/or L-arginine (customized)
Dialyzed fetal bovine serum Cat #26400-044 Invitrogen
The use of dialyzed serum is necessary to avoid the contribution of natural amino acids
present in normal FBS.
The “non-labeled” forms of the aminoacids can be ordered by SIGMA
L-arginine-HCl
L-lysine HCl
Company Cat#
A-6969
L-8662
Stock in PBS
84mg/ml
146mg/ml
Dilution*
* The Dilution is dependent on the cell line. Must be tested by the user.
The labeled Aminoacids can be ordered by Cambridge Isotope Laboratories,
Inc. or Sigma-Isotec
Further cell culture supplements:
Glucose
Antibiotics P/S
Glutamine
Stock
20% in H2O
100x
100x
Dilution
1: 57.14
1:100
1:100
The usage of essential aminoacids to the medium (those not synthesizable by
mammalian cells) forces the cells to use a particular labeled and unlabeled
form. Thus, with each cell doubling the cell population replaces at least half of
the original form of the amino acid. Five cell doublings are enough to achieve
a 100% labelling (incorporation of the specific amino acid).
Differentiation of brown fat cells (see also Project 1 Ronald Kahn).
Differentiation Media: DMEM (see above) + 10% dialyzed FBS +
Stock
Insulin
5,73 mg/ml
Sigma Cat# I5500
1 mM
T3
6.51 µg/ml
Sigma Cat#T-2877
10 µM
Filter sterile (0.2 mcron filter)
Final conc.
0,114 µg/ml
20 nM
0,651 ng/ml
0,01 nM
Dilution
1:50.000
1:10.000
Induction Media:
Differentiation Media +
Dexamethasone
Sigma Cat # D-1756
Indomethacin
Sigma Cat # I-7378
IBMX
Sigma Cat # I5879
Stock in abs. ETOH
2mg/ml
5.1 mM
44,72mg/ml
0,125 M
57.5 mg/ml
250 mM
Final conc.
2 µg/ml
5.1 µM
44,72 µg/ml
0,125 µM
0,115 µg/ml
0,5 mM
Dilution
1:1000
1: 1000
1:500
Differentiation time course and Insulin Induction.
5 min Insulin
induction
Final Insulin concentration for induction: 1µg/ml
Diff. Media
1
2
Ind. Media
4
6
Diff. Media
8
harvest cells
DMEM Media
10
11
12 days
-FBS for 16h
2. Immunopreciptation for large scale Experiment
Lysis buffer:
50mM Tris HCL, pH 7.5, 150mM NaCl , 1% NP-40 , 1mM EDTA ,
0.1% NaDesoxycholate, 1 tablet PIC (Roche cat # 1836145)*, 1mM NaOvan*
*fresh, on day of use
Protein A or G beads for precleaning
Anti-Phosphotyrosine antibody (4G10) agarose conjugated 1mg/ml (50%
slurry), Upstate Cat#16-101
1. Suck off the media completely and add between 700µl to 1ml ice cold
lysis buffer per 15cm dish.
2. Incubate the dishes on ice for 15 min
3. Scrape and collect the cells in tubes (approx. 15-20ml for 10 plates)
4. Incubate on ice for 15min- vortex every 2min
5. Centrifuge with 16000g speed for 15min at 4C
6. Important - save 10-20µl of each state/Exp. for direct lysate and freeze
the lysate at -20C.
7. Transfer the rest into another tube and measure the protein
concentration.
8. According to the Protein concentration mix each state 1:1.
9. Precleaning: For 30-40ml lysate use approx. 1ml µl slurry of Protein A
or G beads, dependent on the antibody
Incubate with gentle rock for 1h at 4C
10. After precleaning centrifuge for 30seconds with 500g and separate the
supernatant fraction. Discard the pellet.
11. Incubate (gentle rocking) the supernatant with 500-700µl slurry of the
agarose conjugated 4G10 antibody for 6h at 4C
12. Separate the 4G10 beads using a Chromatography Column
(Biorad Cat. # 732-1010).
13. Wash the beads with 2x 3ml ice cold lysis buffer
14. Wash with 3x 1.5ml ice cold 50mM Tris pH 7
15. Elute the bound proteins with ice cold 50mM Glycine pH 2.3.
For 700µl beads use 7ml Glycine.
Incubate the beads 3x with 2ml for 2-3min and collect the flow through.
Incubate with 1ml for 2-3min and collect the flow through.
16. To neutralize the flow through add 35µl neutralization buffer per ml flow
through (for 7ml add 245µl NB, NB: 1mM EDTA, 2M Tris-HCl, 1.5M
NaCl adjust to pH 8).
17. Reduce the volume with Centricons (Micon, Centricon YM-3 cut off
3000Da Cat # 4202, see manual) to 20-30µl.
3. In-gel digestion
Buffers
Buffer A: 0.5% Acetic acid
Buffer B: 80% Acetonitril, 0.5% Aceticacid
Buffer A* 5% Acetonitrile, 1%TFA in water
1. Gel Running
After IP (see IP Protocol) apply the protein samples on a NOVEX Gel
Invitrogen Novex Cat.No.: NPO 031BOX
NUPAGE 4-12% BIS-TRIS Gel 1mmx10 well
Running buffer MOPS, 50-60 min, 200V
2. Gel Staining
After PAGE stain with Colloidal Coomassie staining Kit.
Invitrogen, Cat.No.: LC60025, Staining for 4h- o.n.
3. Excision of Protein bands
After staining wash entire gel with water over night. Excise bands of interest
with a clean scalpel cutting as close to the edge of the band as possible
Chop the excised bands into little pieces of 1mmx1mm
Transfer gel pieces into a microcentrifuge tube (1.5ml Eppi)
4. In well Trypsin Digestion. off stained protein samples
Wash gel pieces 2x with 150µl 50% 50mM NH4HCO3 / 50% Ethanol for
20min.
Wash with absolute Ethanol for 20 min for dehydration.
Dry gel pieces with speed vac at room temperature (r.t).
5. Reduction with DTT, Cleaveland´s Reagent Cat. No.: 20290 / Perbio
Add to the dried gel pieces approx 50µl of 10mM DTT in 50mM NH4HCO3 for
60 min at 56C.
6. Alkylation with Iodoacetamid, Cat.No.: I-6125, Sigma
Add 50µl 55mM Iodoacetamid in 50mM NH4HCO3 for 45 min at room
temperature.
Wash with 150 µl 50mM NH4HCO3 for 20 min
Dehydrate with absolute Ethanol 2x for 10 min
Dry pieces in speed vac
7. Digest with trypsin / Cat.No.: V5113, Promega
12.5ng /µl trypsin in 50mM NH4HCO3
Add 10-20µl trypsin and place at room temperature for 10min
Add further 20µl of 50mM NH4HCO3 to keep the gel wet during digestion.
Start digestion by incubating at 37C and digest for 4h or over night.
4. Sample preparation after in Gel digest
1. Transfer the digested proteins (trypsin/ buffer solution) in a new
Eppendorf tube.
2. Add to the gel pieces 100µl 3%TFA/ 30% Acetonitrile and incubate for
20min at r.t. and combine with previous fractions (see 1).
3. Add 70-80µl 3%TFA/ 30% Acetonitrile for 20min to the gel pieces, save
supernatant and combine with fraction from 1 and 2.
4. Add 70-80µl 100% Acetonitrile and combine with previous fractions
5. Add 10-20µl 100% Acetonitrile and combine with previous fractions
6. Reduce the volume of approximately 350µl to 50-70µl in a speed vac
(30-60 min)
7. Add an equal amount (50-70µl) of buffer “A* “ [ 5% Acetonitrile,
1%TFA in water].
5. Preparing and Loading of Stage Tips ( Stop And Go Extraction Tips)
1. Prepare desalting columns as necessary by punching out small disks of
C18 Empore filter using a 22 G flat syringe and plug the disks into
P200 pipette tips. Ensure that the disk is securely wedged in the bottom
of the tip. (Empore disks, 3M, Minneapolis, MN, Cat# 2215)
2. Condition a column by forcing methanol (100µl) through the Empore
disk with a syringe fitted to the end of the pipette tip.
3. Remove any remaining organic solvent in the column by forcing buffer A
through (2x)
4. Force the acidified peptide sample from step 4 through the C18 column.
At this stage the loaded tips can be stored at 4C until used.
5. Wash the column with 20µl buffer A
6. Elute the peptides from the C18 material using 2x10µl buffer B (+0.2%
TFA). Elute directly into an autosampler plate (96wells-plate)
7. Place samples (96wells plate) in SpeedVac and dry down without heat.
Watch carefully – do not overdry keep 1-2µl. This will take approx. 5min.
8. Add 10µl buffer A (+1% TFA).
Literature:
Rappsilber J, Ishihama Y, Mann M. Stop and go extraction tips for matrixassisted laser desorption/ionization, nanoelectrospray, and LC/MS sample
pretreatment in proteomics.
Anal Chem. 2003 Feb 1;75(3):663-70.
5. HPLC system and mass spectrometer.
HPLC:
Agilent 1100 nanoflow system.
Mass spectrometer:
Thermo LTQ-FT - a hybrid linear ion trap-Fourier transform ion cyclotron
resonance MS, Thermo Electron, Bremen Germany.
Reverse-phase nano-LC-MS/MS is performed using an Agilent 1100 nanoflow
LC system (Agilent Technologies Inc.) comprising a solvent degasser, a
nanoflow pump, and a thermostated µ-autosampler. The LC system is
coupled to a 7-Tesla Finnigan linear ion trap-Fourier transform (LTQ-FT)
mass spectrometer, using a modified nanoelectrospray ion source (Proxeon
Biosystems) interface. Binding and chromatographic separation of peptides is
achieved in a 20-cm fused silica emitter (75-µm inner diameter; Proxeon
Biosystems) packed in-house with a methanol slurry of reverse-phase
ReproSil-Pur C18-AQ 3-µm resin (Dr. Maisch GmbH) and mounted in the
nanoelectrospray ion source.
The tryptic peptide mixtures are routinely autosampled at a flow rate of 500
nl/min onto the packed column and then eluted at a flow rate of 250 nl/min. A
linear gradient elution from 95% buffer A (H2O – acetic acid, 100:0.5 vol/vol)
to 50% buffer B (H2O –acetonitrile-acetic acid 20:80:0.5 vol/vol) in 100150min chromatographically separates the peptides.
Literature
Olsen JV, Mann M., Improvevd peptide identification in proteomics by two
consecutive stages of mass spectrometric fragmentation.
Proc Natl Acad Sci U S A. 2004 Sep 14;101(37):13417-22.
Hinsby AM, Olsen JV, Mann M. Tyrosine phosphoproteomics of fibroblast
growth factor signaling: a role for insulin receptor substrate-4.
J Biol Chem. 2004 Nov 5;279(45):46438-47.
6. More Information about SILAC
Please see: http://www.pil.sdu.dk/silac.htm
Literature:
http://www.pil.sdu.dk/silac_bibliography.htm