1. Quantitative proteomics - SILAC Harini Chandra 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.

2. 5 3 2 4 1 Master Layout (Part 1) This animation consists of 2 parts: Part 1: SILAC Part 2: Application of SILAC Light medium Heavy medium Harvest & combine cells Cell lysis & proteolysis Quantification by MS m/z Intensity Mass spectrum Peptide fragments Light Heavy 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.

3. Definitions of the components: Part 1- SILAC 5 3 2 4 1 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.

4. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 1, Step 1: Light medium Heavy medium COO - C HH3NH3N CH 2 CH CH 3 + COO - C DD3ND3N CD2CD2 CDCD CD3CD3 CD3CD3 + Normal/light leucine Deuterated/heavy leucine Cell culture

5. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 1, Step 2: Cell growth & replication L L L L L L L L L L Proteins with light leucine Proteins with heavy leucine

6. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 1, Step 3: Harvest & combine cells 2500 rpm 20 o C 10 min Cultures mixed

7. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 1, Step 4: Cell lysis & proteolytic digestion Lysis buffer Trypsin Intact cells Lysed cells Peptide fragments Heavy Light

8. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 1, Step 5: SDS-PAGE & in-gel digestion Peptide fragments SDS-PAGE Separated peptide bands Peptide fragments with reduced complexity Tubes with buffer

9. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 1, Step 6: LC-MS/MS analysis Peptide fragment HPLC Pump LC Column Sample injector Sample vials Injector Column Column inlet from pump Column outlet to detector Sample Mobile phase Column elution Pump

10. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. Part 1, Step 7: LC-MS/MS analysis Peptide spectrum m/z Intensity Light Heavy m/z Intensity LightHeavy Ratio of peak intensities is indicative of ratio of protein abundance. Quadrupole (scanning mode) Collision cellTOF tubeReflector Detector ESI m3 m1 m4 m2 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.

11. Part 1, Step 8 Action Audio Narration 1 5 3 2 4 Description of the action The MS/MS data analysis shareware has some extra inputs such as 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. As shown in animaion. 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. Your name Search title Email Database(s)SwissProt NCBInr MSDB Enzyme Taxonomy Fixed modifications Variable modification Peptide tol. MS/MS tol. Peptide charge Data file Choose file Monoisotopic Average Proteomics proteomics@gmail.com Sample protein Trypsin Mammalia Bacterial Plant Bacterial Carboxymethyl (C) Oxidation (M) 1.2 Da 0.2 Quantitation Trypsin Chymotrypsin Peptidase # C 13 Data format Instrument Precursor Start search… MALDI-TOF ESI-Q-TOF MALDI-TOF-TOF ESI-Q-TOF MASCOT LC-MS/MS data analysis iTRAQ 4plex SILAC ICAT D8 SILAC

12. 5 3 2 4 1 Master Layout (Part 2) This animation consists of 2 parts: Part 1: SILAC Part 2: Application of SILAC de Godoy, L. M. et al., Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 2008, 455 (7217): 1251-4. Light medium – Normal L-lysine Heavy medium – 13 C 6 / 15 N 2 L-lysine Haploid yeast cells Diploid yeast cells SILAC m/z Intensity Light Heavy Peptide spectrum

13. Definitions of the components: Part 2- Application of SILAC 5 3 2 4 1 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.

14. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. SILAC is a useful quantitative approach that has found applications for several proteomic studies. Part 2, Step 1: Applications of SILAC http://silac.org/applications 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, have been efficiently studied by SILAC coupled with MS. Quantitative proteomic studies using SILAC have been carried out with yeast, which serves as a model organism for eukaryotic cells in providing insights into biological processes. Methylation, which is one of the most common PTMs having various biological roles, has been successfully studied using isotopically labeled methionine residues. One of the more recent applications of SILAC include the identification of protease substrates using differentially labeled bacterial cell cultures. 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. Signaling pathways involving 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. SILAC allows for labeling and monitoring of dynamically changing proteomes of sub-cellular organelles which are involved in several activities during apoptosis in cells.

15. 1 5 3 2 4 ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Part 2, Step 2: Haploid yeast cells Diploid yeast cells Light medium – Normal L-lysine Heavy medium – 13 C 6 / 15 N 2 L-lysine m/z Intensity Light Heavy Peptide spectrum Protein extraction, digestion & LC- MS?MS 97.3% of proteome was found to change less than 50% in abundance between haploid and diploid cells!

16. Interactivity option 1:Step No: 1 (a) 1 2 5 3 4 Unlabelled mouse Normal natural lysine- containing diet 13 C 6 lysine containing diet. Labelled mouse ActionAudio NarrationDescription of the action As shown in the animatio n. 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. 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. Human serum albumin Hemoglobin Average lysine-6 incorporation over 4 weeks 1 2 34 Weeks SILAC ratio 1 2 34 Weeks SILAC ratio

17. Interactivity option 1:Step No: 1 (b) Boundary/limitsInteracativity Type Options Results 1 2 5 3 4 Choose the correct answer. User must choose one of the four options shown above. 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. 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. B) Rate of incorporation of lysine is not calculated properly. C) The lysine incorporation in human serum albumin is defective. D) The long 60-day half life of mouse erythrocytes leads to less labeling of hemoglobin.

18. 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. 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 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: Answers: a) Oxidation of disulphide bonds b) Cleavage at N-terminal of amino acids c) Cleavage at C- terminal 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 c) incorporation of isotope label is 100% d) incorporation of isotope label is 50% 1 5 2 4 3

19. 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