1. 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
Mentor: Dr. Sanjeeva Srivastava
Title of the concept
*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license 1

2. 1 Definitions and Keywords 2 3 4 5
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 MS-based 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.
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. 2 3 4 5 2

3. Learning objectives 1
After interacting with this learning object, the learner will be able to:
Perform culturing of cells using the heavy and light amino acids
Carry out Protein extraction from the cultured cells
Define inoculation of growth in fresh media
Carry out SDS-PAGE gel run
Perform samples through chromatography
Analysis of data and interpretation of the results 2 3 4 5 3

4. 1 Master Layout 2 3 4 5 Slide 5-11 Sample processing Slide 12-16
Sample labelling
Slide 17 3
SDS-PAGE
Slide
18-20
Liquid chromatography 4
Slide
21-23
Data analysis and interpretation 5 4

5. 3 Step 1: 1 2 4 5 T1:Media preparation Audio Narration (if any)‏
Description of the action
Audio Narration (if any)‏
Text to be displayed
Show a measuring balance, with display, ON, OFF and TARE/0 buttons on it. let user ON it, display reading as 0.000g, let user picks up the paper from the rack, makes 1/10 of folding on the sides and places it on the balance. Now the display reading changes to 0.003g. Instruct user to TARE the reading. And animate to click the tare button. Once user clicks it, reading must show ”0”
Measuring balance
When measuring with paper, the weight of the paper need to be tarred from actual reading. 4 5
Video file: Balancing 5 5 5

6. 3 Step 1: 1 2 4 5 T1:Media preparation Audio Narration (if any)‏
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
Clean the laminar air flow thoroughly with ethanol 4 5
Video file: Laminar air flow 6

7. Description of the action/ interactivity
Step 1: 1
T1:Media preparation 2
Description of the action/ interactivity
Audio Narration
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.
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. 3 4 5

8. Description of the action/ interactivity
Step 1: 1
T1:Media preparation 2
Description of the action/ interactivity
Audio Narration
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.
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. 3 4 5

9. Description of the action/ interactivity
Step 1: 1
T1:Media preparation
filter membrane 2
Description of the action/ interactivity
Audio Narration 3
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.
Instruct user to pass the “SILAC medium” through the filter. Please re-draw the filter unit, let user take out 0.22-um 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. 4 5

10. Description of the action/ interactivity
Step 1: 1
T1:Media preparation
autoclave 2
Description of the action/ interactivity
Audio Narration
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.
The media prepared need to autoclaved to be free of contaminants. 3 4 5

11. Description of the action/ interactivity
Step 1:
autoclave 1
T1:Media preparation 2
Description of the action/ interactivity
Audio Narration
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.
Once autoclaved, let the media, cool downs to room temperature to be used for inoculation. 3 4 5

12. 3 Step 2: 1 2 4 5 T2:Inoculation and protein extraction Bacterial
culture 2
SILAC
medium 3
Audio Narration (if any)‏
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.
Place the inoculated flasks in the shaker-incubator at 37’C for 24hrs. 4 5 12

13. Description of the action/ interactivity
Step 1: 1
T1:Media preparation 2
Description of the action/ interactivity
Audio Narration 3
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.
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. 4 5

14. Description of the action/ interactivity
Step 1: 1
T1:Media preparation 2
Description of the action/ interactivity
Audio Narration 3
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.
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. 4 5

15. 3 Step 2: 1 2 4 5 T2:Inoculation and protein extraction
Audio Narration (if any)‏
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.
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. 4 5 15

16. 3 Step 2: 1 2 4 5 T2:Inoculation and protein extraction Day:1 5ml 5ml
Audio Narration (if any)‏
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.
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. 4 5 16

17. 3 Step 3: 1 2 4 5 T3:Protein separation Audio Narration (if any)‏
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 SDS-PAGE 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.
SDS-PAGE sample loading buffer: 0.3 M Tris–HCl, pH 6.8,
10% SDS, 12.5% vol/vol 2-mercaptoethanol, 50% glycerol, 0.016% bromophenol blue Depending upon user set up, one can carry to resolve protein sample through SDS-PAGE followed by in-gel trypsin digestion to carry out MS or directly can do In-solution trypsin digestion followed by MS analysis. 4 5 17

18. 3 Step 4: 1 2 4 5 T4:Liquid chromatography Audio Narration (if any)‏
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.
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. 4 5 18

19. 3 Step 4: 1 2 4 5 T4:Liquid chromatography Audio Narration (if any)‏
Description of the action/ interactivity
For LC-MS setup and parameters details: Mobile phase A – 0.4% acetic acid % 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 bar. 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.
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. 4 5 19

20. 3 Step 4: 1 2 4 5 T4:LC Audio Narration (if any)‏
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.
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. 4 5 20

21. 3 Step 5: 1 2 4 5 T5:Data analysis and interpretation
Heavy region
Light region 3
Audio Narration (if any)‏
Description of the action/ interactivity 4 5 21

22. 3 Step 5: 1 2 4 5 T5:Data analysis and interpretation
Audio Narration (if any)‏
Description of the action/ interactivity 2
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
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. 3 4 5 22

23. 3 Step 5: 1 2 4 5 T5:Data analysis and interpretation
Audio Narration (if any)‏
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.
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. 4 5 23

24. Instructions/ Working area
Slide
12-16
Slide 17
Slide
18-20
Slide
21-23
Slide
1-4
Slide
5-11
Introduction
Tab 01
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Name of the section/stage
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.
Interactivity
area
Button 01
Button 02
Button 03
Instructions/ Working area
Credits 24

25. Questionnaire: APPENDIX 1
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 25

26. Questionnaire: APPENDIX 1
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% 26

27. 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:
Kerner, M. J. et al., Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 2005, 122 (2):
Harsha, H. C., Molina, H. & Pandey, A. Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat. Protoc. 2008, 3:
Websites: 27

28. 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. 28