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1. E 39 Preparing Mouse Embryonic Fibroblasts MEF ssssssssssssseeeeeeeeee een 39 Preparing Matrigel Coated Plates easque p nh Nee quan UP oed n rupe 42 B ffet Rectpessc co dae aoc Seed tede tete ius 43 General Methods for Protein Analysis sss nnt nnne 45 Technical SuppoEbt usos eni tease ede meae ii iesu dn itti 50 Product Qualification eeu Se eee i Se od ee le ee 51 Purchaser Notification neret rmt rient the eiae rera de e e ir edu cornea 52 References anmore e tenet ear TEL doo I LC E M ET C Sob 53 iii iv SILAC Labeling Procedure for Experienced Users Introduction A brief procedure to perform SILAC labeling of human or mouse embryonic stem ES cells is described below For details on media preparation ES cell culture protein analysis and MS analysis refer to the detailed procedure in this manual Step Procedure Prepare cells and medium Perform SILAC labeling 1 Prepare Regular ES Cell and SILAC Heavy and Light ES Cell Medium as follows Regular ES Cell Medium SILAC ES Cell Medium KnockOut D MEM SILAC KnockOut D MEM 20 KnockOut Serum Replacement 20 KnockOut Serum Replacement 2 mM L GlutaMAX I Supplement 100 mg L L Lysine for light medium 0 1 mM MEM Non Essential Amino 100 mg L U PC4 L Lysine Lys for heavy Acids Solution medium 1X 55 uM 2 me
2. Prepare SILAC medium with supplements and normal lysine or isotope labeled lysine 13 1day Prepare MEF conditioned SILAC medium 41 1day Grow ES cells as two different populations grow one ES cell population in MEF conditioned SILAC medium containing light normal lysine and grow the other ES cell population in MEF conditioned SILAC medium containing heavy isotope labeled lysine 17 1day Expand the two ES cell populations for six doubling times to achieve complete incorporation of the labeled amino acid 17 2 weeks Perform cell treatment if needed 21 3 7 days Harvest culture media from each population and mix the media using equal protein amount to identify growth factors OR Harvest cells from each population and mix the cells using a 1 1 ratio based on cell number to identify differential protein expression or unique cell surface markers 23 1day Process the samples using standard protein or peptide separation methods 23 3 5 days Analyze tryptic peptides using MALDI TOF MS or LC MS 27 2 6 days 10 Perform protein identification using MS instrument software or Mascot software suite 30 11 Perform protein quantitation using instrument software such as GPS Explorer 32 7 10 days Continued on next page TM Experimental Overview Continued Experimental Workflow Below is the experim
3. e You need to be familiar with standard stem cell culture techniques using mouse embryonic fibroblasts MEF to successfully use the SILAC Kit If you need help with human or mouse stem cell culture preparation of MEF and conditioned media general protein separation methods refer to published references Bodnar et al 2004 Xu et al 2001 Visit www invitrogen com stemcell for more technical resources on stem cells To obtain easily interpretable results it is important to obtain gt 90 incorporation of the isotope labeled amino acid into proteins You need to determine the efficiency of incorporation as described on page 21 Based on the doubling time of your cell line you can determine the efficiency of incorporation before starting the actual labeling experiment if the doubling time of your cells is 16 18 hours or along with your labeling experiment if the doubling time of your cells is 24 48 hours Greater than 90 incorporation of the isotope labeled lysine into proteins is recommended for SILAC labeling experiments We have routinely achieved gt 95 incorporation using the protocols described in this manual with a variety of cell lines including HeLa 293 and H1 human ES cells Similar incorporation was also observed when labeling a wide variety of cells including yeast and prokaryotes Beynon amp Pratt 2005 TM The SILAC Kits are designed for use by cell biologists and protein biochemists to perform the labe
4. 11 Anesthetize a 13 day pregnant mouse using isoflurane or halothane and perform a cervical dislocation Remove the uterine horns from mouse place in a 100 mm culture dish and wash three times with sterile PBS Cut open embryonic sac with sterile scissors and transfer the embryos to the culture dish Remove the placenta from the embryos and transfer the embryos to a clean 35 mm culture dish Finely mince the embryo gently with scissors add 2 ml trypsin EDTA solution Incubate for 5 10 minutes at 37 C Add 5 ml MEF medium to inactivate trypsin and transfer the contents to a sterile 15 ml centrifuge tube Dissociate MEF cells by trituration Allow large particle to settle to bottom of the tube and transfer supernatant to a T 75 culture flask containing 10 15 ml MEF medium Incubate the flask in a 37 C incubator containing a humidified atmosphere of 5 CO in air When cells are 80 85 confluent 2days passage the cells at a ratio of 1 2 every 3 days Once you have established the MEF freeze a small aliquot of cells Expand the MEF cells for a few passages but not beyond 5 passages Use MEF to prepare the MEF conditioned SILAC medium next page Continued on next page Preparing Mouse Embryonic Fibroblasts MEF Continued Preparing MEF Once the MEF cells reach 80 85 confluency use the cells as feeders to prepare Conditioned MEF conditioned media Perform all steps in a tissue culture hood under sterile R
5. aY Y 48 After SDS PAGE stain the gel with a protein stain to visualize the protein bands Use a Coomassie stain such as SimplyBlue SafeStain for staining or silver stain such as SilverQuest Silver Staining Kit for staining low abundant proteins SimplyBlue SafeStain is a ready to use proprietary Coomassie G 250 stain that is specially formulated for fast sensitive detection and safe non hazardous disposal Proteins stained using the SimplyBlue SafeStain are compatible with mass spectrometry analysis Refer to the manual supplied with stain for protocol details See page vii for ordering information SilverQuest Silver Staining Kit provides a rapid and easy method to silver stain proteins in polyacrylamide gels This kit is specifically designed to provide sensitive silver staining compatible with mass spectrometry analysis The SilverQuest Silver Staining Kit includes destaining solutions that effectively remove silver ions from protein bands in polyacrylamide gels This improves trypsin digestion and subsequent mass spectrometry coverage of the protein as silver ions are known to inhibit trypsin digestion of proteins Chambers et al 1974 Refer to the manual supplied with stain for protocol details See page vii for ordering information Note If you are destaining the gel using the destaining solutions included in the SilverQuest Kit wash the gel piece thoroughly with ultrapure water until the gel piece is com
6. L Arginine 100 mg ml from Step 2 to prepare heavy SILAC ES Cell single labeling medium supplemented with light arginine and heavy lysine Mark the bottle appropriately Store the remaining lysine and arginine solution at 20 C Optional If you are preparing double labeled medium add 0 1 ml Lys 100 mg ml from Step 1 and 0 1 ml Arg 100 mg ml from Step 2 to prepare heavy SILAC ES Cell double labeling medium supplemented with heavy isotope labeled arginine and lysine Mark the bottle appropriately Store the remaining lysine and arginine solution at 20 C Continued on next page 13 Preparing Cells and Media Continued Preparing SILAC ES Cell Medium continued Preparing Regular ES Cell Medium 14 7 To each filter unit add 1 ml 100X L GlutaMAX I 1 ml 100X MEM Non Essential Amino Acids Solution and 0 1 ml 1 000X 2 mercaptoethanol 8 Optional You may supplement the medium with additional growth factors or cytokines if needed for your ES cell line Add 0 1 ml SILAC Phenol Red Solution to each filter unit except when you are planning to analyze secreted proteins avoid adding phenol red as phenol red binds to C18 columns 9 Apply vacuum to each filter unit to filter sterilize each medium 10 To culture ES cells add the following growth factors to each 100 ml medium bottle e Human ES cells human bFGF to a final concentration of 4 8 ng ml e Mouse ES cells Murine LIF to a final concentration
7. Products Continued Additional The table below lists additional products available separately from Invitrogen Reagents For more information visit www invitrogen com or call Technical Support page 50 Product Quantity Catalog no Recombinant Protein G Agarose 5ml 15920 010 SimplyBlue SafeStain 1L LC6060 SilverQuest Silver Staining Kit 1 kit LC6070 NuPAGE Novex 4 12 Bis Tris Gel 10 well 1 0 mm 1 box of 10 gels NPO321BOX NuPAGE MOPS SDS Running Buffer 20X 500 ml NPO0001 NuPAGE MES SDS Buffer 20X 500 ml NP0002 NuPAGE Antioxidant 15 ml NP0005 NuPAGE LDS Sample Buffer 4X 10 ml NP0007 NuPAGE Sample Reducing Agent 10X 250 ul NP0004 XCell SureLock Mini Cell 1 unit EI0001 Max Ion Peptide MALDI Matrix Kit 1 kit MS10005 Invitrosol LC MS Protein Solubilizer 5X 5 ml MS10007 Invitrosol MALDI Protein Solubilizer Kit 1 kit MS10001 1 D PAGE Cleavable ICAT Reagent Application Kit 1 kit MS10012 Cleavable ICAT 10 assay Kit 1 kit MS10022 Cleavable ICAT Bulk Reagent Kit 1 kit MS10010 iTRAQ Reagents Methods Development Kit 1 kit MS10013 iTRAQ Reagents MultiPlex Kit 1 kit MS10016 Trypsin 1 kit MS10015 Antibodies A large variety of high quality antibodies against various proteins is available from viii Invitrogen Visit our website for details or contact Technical Support page 50 Overview Introduction SILAC Technology Introduction The SILAC Stable Iso
8. SILAC Phosphoprotein Identification ID and Quantitation Kit with U Cg L Lysine Lys and D MEM 1 kit SP10001 with U Cq L Lysine Lys and RPMI 1640 1 kit SP10005 SILAC Membrane Protein Identification and Quantitation Kit with U C4 L Lysine Lys and D MEM 1kit SM10002 with U C4 L Lysine Lys and RPMI 1640 1 kit SM10006 Media The table below lists cell culture media products available separately For more Components information visit www invitrogen com or call Technical Support page 50 Product Quantity Catalog no KnockOut D MEM 500 ml 10829 018 KnockOut Serum Replacement 500 ml 10828 028 GlutaMAX I Supplement 100 ml 35050 061 MEM Non Essential Amino Acids 10 mM 100X Liquid 100 ml 11140 050 2 mercaptoethanol 1 000X liquid 50 ml 21985 023 D MEM high glucose 1X liquid 500 ml 11965 092 Fetal Bovine Serum FBS ES Cell Qualified US 100 ml 16141 061 L Glutamine 100X 200 mM 20 ml 25030 149 Collagenase Type IV 1g 17104 019 bFGF basic Fibroblast Growth Factor Human Recombinant 10 ug 13256 029 Trypan Blue Stain 100 ml 15250 061 Phosphate Buffered Saline PBS 1X 500 ml 10010 023 Recovery Cell Culture Freezing Medium 50 ml 12648 010 TrypLE Express Stable Trypsin Replacement Enzyme 1X without 100 ml 12604 013 Phenol Red 500 ml 12604 021 TrypLE Express Stable Trypsin Replacement Enzyme 1X with 100 ml 12605 010 Phenol Red 500 ml 12605 028 Continued on next page vil Accessory
9. blade to excise the gel bands Be sure to use polypropylene microcentrifuge tubes and HPLC grade solvents Some manufacturers use mold release agents in manufacture of some plastics Avoid using such plasticware Ensure the MS instrument was properly tuned and calibrated prior to sample analysis Ensure the chromatographic separation was effective and the columns used for separation were free of contaminants from prior separations Always run blanks between chromatographic separations to avoid any contaminations Use clean MALDI plates for analysis Continued on next page 37 Troubleshooting Continued Observe 2 peaks for proline containing peptides Conversion of arginine to proline Inconclusive identification and quantitation of phosphoproteins due to poor data quality Loss of phosphorylation No phosphoprotein enrichment step performed Lower incorporation Incomplete incorporation of heavy amino acid efficiency Introduced exogenous sources of light amino acid that can reduce total labeling 38 When proline containing peptides are used for quantitation the correction factor in peak intensity for total heavy labeled peptides should be the sum of peak intensities of these mass channels Liang et al 2006a To avoid loss of phosphorylation process the samples immediately after collection and perform all steps at 4 C Do not store the samples for prolonged periods of time Use lysis buf
10. factor B Sc gil38372433 102577 5 5 3200 KIAA0731 protein Homo sapien gi3882183 1228640 9 1600 ATP dependent RNA helicase A gil3315658 140788 0 5 3500 annm PROTEIN SUMMARY MALDI PLATES SOURCEPLATES METRICPLOT METRICDATA MSMS SUMMARY SPECTRUM PEAK TABLE afro fa 1 gil 5453832 Mass 111266 Total score 408 Peptides matched 17 oxygen regulated protein precursor oxygen regulated protein 150kD Homo sapiens Check to include this hit in error tolerant search or archive report Query Observed Mr expt Mr calc Delta Miss Score Rank Peptide V 23 1005 51 1004 50 1004 54 0 05 0 36 1 YFQHLLGK W 47 1011 54 1010 53 1010 54 0 01 0 as 1 YFQHLLGK Lys heavy W 48 1011 55 1010 54 1010 54 0 00 0 35 1 YFQHLLGK Lys heavy W 49 1012 54 1011 53 1011 55 0 01 0 41 1 EVQYLLNK Lys heavy F 50 1012 56 1011 55 1011 55 0 00 0 as 1 EVQYLLNK Lys heavy 92 1029 48 1028 47 1028 48 0 01 1 7 3 KYPDYESK 132 1041 49 1040 48 1040 48 0 00 1 15 4 KYPDYESK 2 Lys heavy 658 1250 60 1249 60 1249 59 0 01 0 79 1 FFGDSAASMAIK Lys heavy 745 1295 78 1294 78 1294 79 0 01 0 34 1 LPATEKPVLLSK 884 1372 69 1371 68 1371 68 0 00 6 at 1 EVEEEPGIHSLK Lys heavy 885 1372 69 1371 69 1371 68 0 01 0 4 EVEEEPGIHSLK Lys heavy arno an arna cn aran 32 0 n2 a arncnecvananceey iffstart l te li amp searchresults Paint E GPs Explorer Tm
11. future use Prepare the MEF conditioned SILAC light and heavy labeled media SU m co qus p e MEF medium D MEM FBS and L glutamine are available from Invitrogen page vii medium recipe is on the next page e Light and heavy labeled SILAC media page 13 e bFGF page vii e Trypsin page vii e Appropriate tissue culture plastic ware and culture dishes e 37 C incubator with a humidified atmosphere of 5 CO in air e Sterile centrifuge tubes e Reagents to determine viable and total cell counts page vii e 13 day pregnant mouse e 1X PBS page vii Continued on next page 39 Preparing Mouse Embryonic Fibroblasts MEF Continued Preparing MEF Medium Harvesting MEF 40 Prepare the MEF medium containing 10 FBS and supplemented with L Glutamine using basal D MEM as described below Perform all steps in a tissue culture hood under sterile conditions 1 2 3 4 Remove 100 ml D MEM from 1 L D MEM bottle and replace with 100 ml FBS To each 1 L medium bottle add 10 ml 100X 200 mM L Glutamine Optional Add 10 ml 100X Penicillin Streptomycin page vii if needed highly recommended Filter sterilize the medium using 0 22 um filtration device Store the medium at 2 to 8 C protected from light until use The medium is stable for 6 months when properly stored avoid introducing any contamination into the medium Perform all steps in a tissue culture hood under sterile conditions 1 10
12. isotopically labeled amino acids Bae amp Chen 2004 Sechi amp Oda 2003 Zhu et al 2002 The SILAC technology is a result of further developments of this method by Mathias Mann Ong et al 2002 using stable isotopic amino acids as labels in cell culture which when combined with global differential MS analysis provides a tool to identify and quantitate proteins in complex biological samples TM In SILAC experiments two ES cell populations are grown in identical cell culture media deficient in one or more essential amino acids One cell population is grown in medium with heavy isotopic amino acid while the other cell population is grown in medium with light normal amino acid The natural metabolic machinery of the cells is utilized to label all cellular proteins with the heavy amino acid Amanchy et al 2005 After trypsin digestion the peptides containing the light or heavy amino acids are chemically identical and can be processed together using any protein separation method eliminating quantitation errors due to unequal sampling Because the peptides are isotopically distinct they can be easily distinguished by mass using MS analysis Based on the relative peak intensity of the isotopic peptide pairs you can quantitate differential protein expression and identify differential post translational modifications between samples Liang et al 2006b or in response to a stimulus Liang et al 2006a For a system overview see
13. minutes to detach the ES cells Note The incubation time varies and you need to monitor the detachment under the microscope When majority of colonies are detached or the colony edges are rounded you can proceed to the next step Add 1 ml of the appropriate MEF conditioned SILAC Light or Heavy ES Cell Medium to each well of a 6 well plate Transfer the ES cell suspension to sterile 15 ml centrifuge tubes and centrifuge at 200 x g for 5 minutes Remove the media and resuspend the colonies in the appropriate volume of MEF conditioned SILAC Light and Heavy ES Cell medium in two separate sterile 15 ml conical tubes Triturate the cells into small clusters of 50 500 cells but not to a single cell suspension Determine the viable and total cell count on an aliquot of ES cells using the trypan blue exclusion method Proceed immediately to Labeling and Cell Culture next page Continued on next page 19 Isotopic Labeling in Cell Culture continued Labeling and Cell Culture 20 Instructions for performing labeling with Lys are described below 1 TM Transfer the ES cells as follows into 35 mm MatriGel coated plates to obtain a seeding density of 5 x 10 1 5 x 10 cells cm cell culture plate e Light Cell Population Transfer the ES cells in 4 ml MEF conditioned SILAC medium containing light lysine prepared as described on pages 13 and 41 e Heavy Cell Population Transfer the ES cells in 4 ml MEF condit
14. or TFA over 5 minutes Note Use 0 1 formic acid solution on ESI based instruments and 0 1 TFA solution on off line LC MS MS analysis using MALDI TOF TOF e For a complex sample use a gradient of 5 45 v v acetonitrile in 0 1 formic acid or TFA over 90 minutes or up to 120 minutes and then use a gradient of 45 95 acetonitrile in 0 1 formic acid or TFA over 30 minutes or up to 60 minutes 29 Protein Identification and Quantitation Introduction Important Note Protein Identification 30 Once MS MS analysis is complete perform protein identification and quantitation as described in this section This section is designed for experienced users of MALDI TOF and LC MS analysis especially core facility personnel that are familiar with standard techniques and instruments for MS analysis e Be sure to always compare the results with cells grown in the light and heavy medium containing each amino acid at the same concentration e The screen shots included in this section are provided as guidelines and may not represent the exact screen that you may view for the software if the software has been upgraded These screen shots were captured using GPS Explorer 3 x software Protein identification is performed by searching the peptide fragments obtained after MS MS analysis against non redundant protein databases Most of the MS instruments are supplied with software that is capable of protein identification You may u
15. page 3 For details on light and heavy amino acids see page 17 Continued on next page Overview Continued SILAC Kit Note Advantages The SILAC Stem Cells Protein Identification and Quantitation Media Kit allows identification of key growth factors that regulate proliferation and differentiation as well as analysis of signal transduction For detailed description on each kit component see page 4 Conventionally ES cells are maintained on a layer of growth arrested mouse embryonic fibroblasts MEF However it has been shown that pluripotent ES cells can be maintained in defined medium that is conditioned by prior exposure to the MEF feeder layer Xu et al 2001 The SILAC Stem Cell Kit is designed to provide gt 90 incorporation of labeled amino acids into ES cells maintained on MEF conditioned medium see Note below and the labeling does not affect the growth or pluripotency of ES cells We have routinely achieved gt 95 incorporation using protocols described in this manual with a variety of cell lines including HeLa 293 and H1 human ES cells Similar incorporation was also observed when labeling a wide variety of cells including yeast and prokaryotes Beynon amp Pratt 2005 However lower incorporation efficiencies maybe due to unequal sampling or the introduction of exogenous light label amino acid into samples during the protocol See page 19 for recommendations to avoid introducing exogenous light labe
16. peptides to dryness after trypsin digestion trypsin solution If the peptides are concentrated to dryness the peptides are difficult to resuspend resulting in loss of peptides Abundant proteins are Sample is too complex for Enrich the subcellular fraction of interest well identified and the current analytical quantitated but the scheme protein of interest is below background Enrich for specific peptides phosphopeptides glycopeptides or N terminal peptides of interest Perform an additional dimension of LC or longer gradient on some protein bands of interest Peaks observed for Incomplete incorporation Perform the labeling for at least 6 doublings to unlabeled light amino of heavy amino acid ensure complete incorporation of the label Be sure acid for the protein to use log phase with gt 90 viability TM Additional supplements Always use KnockOut Serum Replacement to added to the medium may prepare the medium Do not use regular FBS or contain amino acids use any other media supplements that may contain free amino acids Amino acid prepared in Prepare the amino acid using basal complete medium unsupplemented KnockOut D MEM supplied with the kit as described on page 13 Do not use any other complete medium to prepare the amino acids See Note on page 19 Continued on next page 36 Troubleshooting Continued The relative abundance between most of the heavy to light labeled proteins is not
17. soft ATD 2 32AM Continued on next page 34 Protein Identification and Quantitation Continued Interpreting the To analyze differential protein expression results with SILAC experiments Results review the data in the column Avg ICAT Ratio H L as shown in the example TM of GPS Explorer results indicated with a circle on the previous page The ratio indicates up regulation or down regulation for various proteins analyzed For example the protein oxygen regulated protein precursor shows an Avg ICAT Ratio H L of 1 76 suggesting that this protein may be up regulated while the protein bA462D18 3 2 ribosome binding protein shows a ratio of 0 746 suggesting that this protein may be down regulated An Avg ICAT Ratio H L ratio of zero indicates the following e Only 1 peak from the peak pair was identified and therefore the software was unable to calculate a ratio This happens when the signal for the peptide is very low for low abundant peptides and is sometimes occluded by background or if the ratio of peak pair relative abundance is very high gt 10 or very low 0 3 For such peptides it is important to go back to the original raw data file and confirm manually e The cell treatment may have lowered the expression of the peptide in treated cells such that the software is unable to identify the signal as a significant signal The results are significant if the coefficient of variance CV is 3076 A significan
18. such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corp 1600 Faraday Ave Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com Use of this product according to the method provided in the product literature is the subject of one or more of U S patent numbers 6 642 059 and 6 391 649 owned by the Rockefeller University and licensed to Invitrogen Corporation The purchase of this product conveys to the buyer the non transferable license to use the purchased amount of the product and components of the product in internal research conducted by the buyer whether the buyer is an academic or for profit entity subject to certain restrictions as fol
19. the set specifications The GlutaMAX I Supplement is qualified by performing tests for pH osmolality and is tested for the absence of bacterial and fungal contaminants Product must meet the set specifications The MEM Non Essential Amino Acids Solution is qualified by performing tests for pH and is tested for the absence of bacterial and fungal contaminants Product must meet the set specifications The 2 mercaptoethanol is qualified by performance testing performing tests for pH osmolality and is tested for the absence of bacterial and fungal contaminants Product must meet the set specifications The SILAC Amino Acids L Lysine isotope labeled L Lysine and L Arginine must meet the following specifications L isomer gt 99 BC atom 97 Endotoxins Low Sterility Sterile with no bacterial or fungal contaminants In addition the degree of incorporation achieved with each lot of amino acids is confirmed by performing the labeling experiment as described in this manual After labeling the cells are lysed and the lysate is analyzed by SDS PAGE At least 3 intense protein bands are subjected to in gel trypsin digestion followed by MALDI TOF MS analysis The observed peak pairs must be separated by the correct mass difference that is proportional to the theoretical isotopic shift the intensity of the peak pairs is 1 1 and the incorporation of the heavy label is gt 90 51 Purchaser Notification Limited Use Label Licen
20. 1 1 Poor amino acid incorporation more apparent when labeling with lysine Sequence database search identifies keratin as the top candidate MS spectra contaminated with peaks at regular interval e g 44 Da repeats of polyethylene glycol The Coefficient of Variance CV for the protein quantitation within one experiment exceeds 30 Error in mixing cells or determining protein amount Arginine terminating peptides cause ionization suppression effects that impair the detection of lysine terminating peaks in MALDI Samples contaminated with keratin Samples contaminated by polymer NP 40 Triton X 100 Improper MS analysis Count the cells prior to mixing and adjust the number of cells harvested to ensure the cells from two populations are mixed in a 1 1 ratio by cell number or equal protein amount Be sure to use log phase cells with gt 90 viability Cell treatment may cause change in morphology resulting in an error in cell number In this case mix the cells using equal protein amount Perform analysis using LC MS or include a simple fractionation step with a ZipTip with Cis resin use only a 30 elution step prior to MALDI MS analysis Always wear gloves while handling the gels and use ultrapure proteomics grade reagents for in gel trypsin digestion Prepare fresh buffers Perform all gel manipulations in a clean dust free environment away from a door or window and always use a fresh razor
21. 47 54 1046 53 1046 55 0 02 0 23 1 LAGLFHEQR Iv 174 1053 55 1052 54 1052 55 0 01 0 70 1 LAGLFNEQR Arg heavy 175 1053 56 1052 55 1052 55 0 00 0 19 9 LAGLFNEQR Arg heavy Iv 176 1053 56 1052 55 1052 55 0 00 0 39 1 LAGLFNEQR Arg heavy 178 1053 56 1052 56 1052 55 0 00 0 9 6 LAGLFNEQR Arg heavy M 352 1124 58 1123 58 1123 58 0 00 0 29 1 TLGGLEMELR Arg_heavy M 353 1124 59 1123 58 1123 58 0 00 0 39 1 TLGGLEMELR Arg heavy M 56 1250 60 1249 60 1249 59 0 01 0 79 1 FFGDSAASMAIK Lys heavy Mw 708 1276 66 1275 65 1275 65 0 00 0 39 1 EAGMQPQLQIR Arg heavy Once protein identification is complete using Mascot or other instrument specific software perform quantitation for differential protein expression Protein quantitation is performed using the software available with your MS instrument such as GPS Explorer software See page 10 for a list of MS instrument software packages for SILAC data analysis GPS Explorer 3 0 software is innovative applications software that supports many biological workflows such as traditional in gel digestion MDLC MS MS LC MALDI and PTM discovery all with intelligent results dependent analysis using RDA software feature Currently only one pair of light and heavy Lys or Arg at a time can be selected for quantitation using GPS Explorer software Continued on next page Protein Identification and Quantitation Continued Using GPS Brief instructions are provided below to set up the G
22. 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_service invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Limited Warranty 50 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service please contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to the specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable
23. 8 Peter 2000 Simpson 2003 Speicher 2004 e Sample concentration of 200 500 nM in a total volume of 5 ul e Prepare samples preferably in ultrapure water methanol or acetonitrile e Sample must contain 10 mM buffer or salts e Use Max Ion Peptide MALDI Kit for MALDI TOF MS analysis next page The tryptic peptides page 49 can be analyzed using the following MS analysis methods Important For identifying and quantitating proteins using SILAC technology it is important to perform MS analysis using tandem MS instruments that are capable of performing MS MS analysis e For samples of low complexity use MS fingerprinting techniques to identify and quantitate proteins e For samples of moderate complexity use MALDI TOF MS analysis We routinely use 4700 Proteomics Analyzer MALDI TOF TOF instrument from Applied Biosystems Other instruments such as Bruker Reflex III Bruker Daltonics or Voyager DE STR MALDI TOF Workstation Applied Biosystems are also suitable e For complex samples use on line or off line LC MS MS or two dimensional LC MS MS You may use Micromass Q Tof Premier Mass Spectrometer Waters or OSTAR Pulsar quadrupole TOF tandem MS Applied Biosystems equipped with a nanoelectrospray ion source or off line separations followed by MALDI TOF TOF analysis Some recommended gradients for LC MS are listed on page 29 Continued on next page 27 Mass Spectrometric Analysis Continued MS Reagents
24. AC technology you will e Grow your human or mouse embryonic stem ES cells as two different populations e Metabolically label one ES cell population using non radioactive isotopic labeled essential amino acid heavy amino acid while labeling the second ES cell population using normal essential amino acid light amino acid during cell culture e Harvest ES cells or medium from each population after the isotopic labeled amino acid is incorporated into the cellular proteins usually complete incorporation is achieved within six doublings e Mix the cells or medium from each population e Process the samples using standard protein or peptide separation methods e Analyze tryptic peptides or phosphopeptides by MS analysis e Perform protein identification and quantification TM The SILAC Kits are designed for cell labeling experiments performed by cell biologists and protein biochemists while working with a proteomics core facility for sample processing and MS analysis You need to identify a proteomics core facility capable of identifying proteins from Coomassie or silver stained gel bands for MS analysis Review the information on page 9 before starting the labeling experiments This manual provides the following information e Basic information for preparing cell culture media and growing cells e Performing isotopic labeling of cells e Guidelines for preparing cell lysates and processing the lysates for analysis e Guideline
25. General methods for protein analysis after labeling your ES cells with SILAC heavy and light media are described in this section If you have established protein analysis methods for ES cells in your laboratory use the established methods Perform immunoprecipitation if needed Process the lysate or immunoprecipitated proteins using SDS PAGE Stain the SDS PAGE gel using Coomassie or silver staining Excise the bands of interest from the gel or cut the gel into 40 equal pieces Samo mr Perform in gel trypsin digestion To obtain the best results we recommend using NuPAGE Novex Bis Tris Gels You may use Novex 4 20 Tris Glycine Gel or any other SDS PAGE gel of choice for performing SDS PAGE Use an appropriate percentage of acrylamide gel that best resolves your proteins of interest Due to the large variety of antibodies that can be used for immunoprecipitation it is not possible to have a single immunoprecipitation protocol that is suitable for all antibodies Use the immunoprecipitation procedure from this section as a starting protocol and based on your initial results optimize the protocol by changing the antibody concentration buffer formulation and incubation time If you have an optimized immunoprecipitation protocol for a specific antibody use the optimized protocol You need the following items Ordering information is on page vii e NuPAGE Novex Bis Tris Gel e NuPAGE MES MOPS SDS Running Buffer e NuPAGE
26. Instrument ions series definitions oracle Installation Products gt 4 Search Form MS MS 4 Mascot configuration file E3 symantec Client Security gt amp Search Form Peptide Mass Fingerprint gt VERITAS NetBackup gt Search Form Sequence Query 8 amp winzip gt amp Search log 7 Acrobat Reader 5 0 Search status 4B Internet Explorer C3 uninstaliShield Al Outlook Express ga n eeu Add the following text at the end of the Mascot Modification File to enable identification of proteins and isotopic peptide pairs for SILAC Title Lys light Residues K 128 09497 128 1741 if stat 3 EEE ees Explorer TM Softwer S untited Paint Title Lys_heavy Residues K 134 09497 134 1741 Title Arg_light Residues R 156 10112 156 1875 Title Arg_heavy Residues R 166 10112 166 1875 This will show isotope labeled lysine heavy lysine 6 Da larger than normal lysine light lysine and isotope labeled arginine heavy arginine 10 Da larger than normal arginine light arginine Set the mass tolerance of the precursor peptide ion at 200 ppm and mass tolerance for the MS MS fragment ions at 0 5 Da Select the variables modification in the setting for data analysis as follows depending on the type of labeling experiment e Fora single label experiment with Lys select a pair of light and heavy Lys as variables e Fora double label experiment with Lys and Arg select a pair of l
27. Invitrogen SILAC Stem Cells Protein Identification ID and Quantitation Media Kit For identifying and quantifying proteins from embryonic stem cells Catalog no MS10036 Version A 17 November 2006 25 0982 ii Table of Contents Table of Contents 22 eee reete tee n tee re ate epe eee e AE ee iii SILAC Labeling Procedure for Experienced Users sse V Kit Contents and Storage ees sitate in rire eee abend atii ded e n eet vi Accessory Products e e iUe UBER GU ee utet e tee s vii MTRO CU C TON 1 OVERVIEW ik A EE E gula denda ce tide Head dm n gud ue bn Geeta de 1 Description of KIECOMPOnentS siai E E E e EE ERER ane EE tenete netten tenete neis 4 Experimental Overview eie eene e ee TEE SER e E AE E a e a en 6 MethodS eaisievactes oct sheesh statue de suedaredeeshoushcvadediecsitencuustesvesavtsinns aluassavantshedssteestiaducnechuiduenidodtedsbesetnnces 9 Before Starting eene Gedeon edt iem Gnd de ird e etta di Testes eH 9 Preparing Cells and Media nennen 11 Isotopic Labeling ir Cell Cult re bee edente e t ee a erts 17 Processing Samples eco us ceases ahs sea cess bee eme dedi ge Ma OI p br RO HR REM ROS 23 Mass Spectrometric Analysis i22 saei ea eiie n a nu eA aie de re de ee EE 27 Protein Identification and Quantitation sess ener eterne nne t nennen ensi nn nn 30 Troubleshooting sn ostendit ede iios itte edited ovii tee ied ie on ien ters in e irs 36 pIcnnpemee
28. LAC Phenol Red Solution 10 g L optional e 2 mercaptoethanol 1 000X e MEM Non Essential Amino Acids Solution 10 mM 100X e U C L Lysine HCl Lys To culture human or mouse ES cells you need to prepare the following media e SILAC ES Cell Medium see next page for recipe Components to prepare the SILAC ES Cell Medium are supplied with the SILAC Stem Cells Kit Use this medium for labeling experiments only Do not use this medium for routine maintenance of ES cells e Regular ES Cell Medium see page 14 for recipe Components to prepare the Regular ES Cell Medium are not supplied with the kit You need to purchase the media components separately from Invitrogen page vii Use this medium for routine maintenance of ES cells Do not use this medium for SILAC labeling experiments Continued on next page Preparing Cells and Media Continued Preparing SILAC Prepare the SILAC ES Cell Medium 100 ml each using the components ES Cell Medium supplied in the kit as described below Adjust the reagent volumes accordingly to prepare gt 100 ml medium To prepare Regular ES Cell Medium see next page Perform all steps in a tissue culture hood under sterile conditions SILAC KnockOut D MEM 20 KnockOut Serum Replacement 100 mg L L Lysine 100 mg L L Arginine 2 mM L GlutaMAX I Supplement 0 1 mM MEM Non Essential Amino Acids Solution 1X 55 uM 2 mercaptoethanol 4 8 ng ml human bFGF human ES c
29. Matrix Kit The Max Ion Peptide MALDI Matrix Kit is designed for peptide analysis by using MALDI TOF MS The kit includes a proprietary resin that promotes the formation of homogeneous thin films of matrix crystals improves the ionization of analyte species and suppresses salt effects resulting in improved spectral quality enhanced signal to noise and reduced matrix background Depending on the type of MS instrument that you have you may be able to e Perform fully automated analysis of SILAC raw data This is supported through the MS instrument software for protein identification and quantitation see page 10 for a list of instrument software packages OR e Perform semi automated analysis of SILAC raw data This is supported through the MS instrument software for protein identification but you need to perform protein quantitation using manual calculations contact Technical Support page 50 or consult the instrument vendor for detail Continued on next page Mass Spectrometric Analysis Continued Recommended If you are using LC MS analysis the following gradients are recommended If Gradients for LC you optimized the LC MS analysis with specific gradients that are suitable for MS your analysis use the optimized gradients for your analysis e For samples with less complexity use a gradient of 5 45 v v acetonitrile in 0 176 formic acid or TFA over 45 minutes and then use a gradient 45 95 acetonitrile in 0 1 formic acid
30. Medium Maintain ES cells at a relatively high density and passage ES cells when they are 70 80 confluent i e tightly packed intermediate size colonies close to each other but not touching each other Passage the ES cells at a ratio of 1 3 to 1 6 4 7 days but change the medium every day After the ES cells are established be sure to freeze an aliquot of cells for future use Follow the manufacturer s recommendations for preparing the freezing medium and freezing cells After thawing allow the ES cells to undergo at least 4 doublings in MEF conditioned Regular ES Cell Medium prior to using the ES cells for SILAC labeling Keep the ES cell passage number low Once you have established the human or mouse ES cell culture on MEF conditioned medium and frozen an aliquot of cells you are ready to perform SILAC labeling as described on page 17 See below for the recommended number of cells needed for labeling You need log phase ES cells with 90 viability to perform successful labeling Based on the type of analysis that you wish to perform the approximate number of ES cells needed for each analysis from each cell population are listed below 60 x 10 or cells from 2 x 100 mm plates for identifying secreted growth factors 30 x 10 or cells from 1 x 100 mm plate for studying differential protein expression 107 108 or cells from 2 to 3 x 100 mm plate for studying signal transduction pathway 10 cells to determine the efficie
31. Note 28 A variety of reagents for MS analysis is available from Invitrogen see page vii for ordering information Invitrosol LC MS Protein Solubilizer The Invitrosol LC MS Protein Solubilizer is a novel surfactant blend that maintains a variety of hydrophobic proteins in solution does not interfere with protease activity and is compatible with reverse phase high pressure liquid chromatography RP HPLC and LC coupled electrospray ionization mass spectrometry ESI MS separations of the tryptic digested peptides Use Invitrosol LC MS Protein Solubilizer to remove incompatible buffer components prior to MS analysis or during in gel trypsin digestion to improve the solubility of hydrophobic tryptic peptides Invitrosol MALDI Protein Solubilizer Kit TM The Invitrosol MALDI Protein Solubilizer Kit is specifically designed for direct MALDI TOF MS analysis of hydrophilic or hydrophobic intact proteins and peptides eliminating the need for solid phase extraction acid hydrolysis and matrix crystal washing The Invitrosol MALDI Protein Solubilizer A and B are ready to use reagents composed of unique proprietary detergent formulations that are designed to minimize suppression effects on the ionization of peptides intact proteins and minimize cluster formation and effectively solubilize hydrophobic proteins and improves sequence coverage of tryptic peptides in solution without affecting the sensitivity Max Ion Peptide MALDI
32. NuPAGE LDS Sample Buffer and then add 20 ul NuPAGE Reducing Agent 10X Heat the samples at 70 C for 8 10 minutes Load the samples from light and heavy medium side by side without mixing on a NuPAGE Novex 4 12 Bis Tris Gel and perform electrophoresis using NuPAGE Novex MES or MOPS SDS Running Buffer Be sure to load appropriate protein standards on the gel Important Avoid any sample cross contamination and always wear laboratory gloves and work in a laminar hood Stain the gel with Coomassie R 250 Stain Note You may transfer the gel to the core facility to perform trypsin digestion and MS analysis For more information on proteomics core facilities that offer MS analysis for SILAC see page 9 Excise 3 4 side by side intense protein bands from each lane Perform in gel trypsin digestion page 49 Perform MALDI TOF MS analysis page 27 See next page for expected results If you do not obtain gt 90 incorporation see Troubleshooting page 36 Continued on next page 21 Isotopic Labeling in Cell Culture Continued Example of An example of results obtained after determining the efficiency of incorporation Results is shown below The MS analysis should show an increase in mass by 6 daltons for peptides labeled with Lys when compared to peptides labeled with normal Lys see figure below Note If you have used double labeling with Arg and Lys then the MS analysis should show an increase in mass by 6 and 10
33. PS Explorer software for Explorer for SILAC data analysis For details on using the software follow the manufacturer s Protein instructions Quantitation 1 Start GPS Explorer software on the MS instrument AB MDS Sciex Family of MALDI TOF TOF Analyzers 2 Navigate to the Data Analysis screen 3 Select the following as variable modifications in the Analysis Settings Screen as shownin the figure below e Fora single label experiment with Lys select a pair of light and heavy Lys as variables Ensure the mass difference of 6 Da shows up at the bottom of the screen in ICAT Delta Mass under ICAT Settings e Fora double label experiment with Lys and Arg select a pair of light and heavy Lys or a pair of light and heavy Arg Simultaneous selection of both labeled Lys and Arg does not work Ensure the following mass difference shows up at the bottom of the screen in ICAT Delta Mass under ICAT Settings indicated with an arrow in the figure below e 6Damass difference when Lys is selected or e 10 Da mass difference when Arg is selected 4 Makesure the ICAT Quantification box is checked and the ICAT Pair Tolerance is set to 150 ppm under ICAT Settings see figure below Analysis Settings Originated from Analysis Settings Template Open Analysis Template Save Analysis Template As RDA Precursor Instrument Acquisition Processing MS MS Peak Filtering Database Search Template Originated from Template D
34. Sample Reducing Agent 10X e NuPAGE LDS Sample Buffer 4X and NuPAGE Antioxidant e XCell SureLock Mini Cell for electrophoresis of the gel e Sterile tubes e Antibody Protein A or Protein G Agarose for immunoprecipitation if needed e Sequencing grade trypsin 10 ng ul dissolved in 25 mM ammonium bicarbonate pH 8 0 store on ice until use e 25 mM ammonium bicarbonate buffer pH 8 0 for trypsin digestion e 5 formic acid FA e 10096 and 70 v v acetonitrile Continued on next page 45 General Methods for Protein Analysis Continued Immuno Immunoprecipitation protocol using Protein G Agarose is described below You precipitation may use Protein A beads if desired 1 To the cell lysate prepared as described on pages 24 26 add 15 ul Protein G Agarose slurry 50 slurry in lysis buffer per 1 ml lysate to pre clear the lysate Rock the lysate at 4 C for 1 hour Centrifuge at 10 000 x g for 1 minute at 4 C Transfer the supernatant to a sterile tube and place on ice Qu sc p Add 50 100 ug of the antibody against the protein of interest Note You may optimize the amount of antibody used based on the initial results 6 Iftheantibody is already coupled to Protein A or Protein G agarose proceed to Step 8 directly Add 100 ul of the Protein G Agarose slurry to the supernatant Rock for 8 16 hours at 4 C Centrifuge at 10 000 x g for 5 minutes at 4 C Remove the supernatant 10 Wash the agarose pellet
35. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Product Qualification Introduction SILAC KnockOut D MEM Media KnockOut Serum Replacement GlutaMAX I Supplement Amino Acids Solution 2 mercaptoethanol SILAC Amino Acids The components of the SILAC Stem Cells Protein ID and Quantitation Media Kit is qualified as described below For details on the qualification see the Certificate of Analysis shipped with the product or available at www invitrogen com The SILAC media is tested for pH osmolality endotoxin and is tested for the absence of bacterial or fungal contaminants Product must meet the set specifications Product performance is confirmed by evaluation of its ability to support the growth of undifferentiated D3 ES cell colonies on inactivated mouse embryonic fibroblasts The product is also tested for pH osmolality endotoxin and is tested for the absence of bacterial or fungal contaminants Product must meet
36. better than Lys containing peptides in MALDI MS resulting in better sensitivity and sequence coverage Using double labeling increases the number of informative peptides making the method more sensitive Use U C L Arginine and U C L Lysine for routine quantitative protein analysis Use U 9C 5N4 L Arginine and U C L Lysine for quantitative protein analysis when a higher level of confidence is required in the identification Experimental Overview The flow chart for the experimental outline using the SILAC kit is shown below Flow Chart See next page for an experimental outline Prepare Media Grow two cell populations With light With heavy Lys and light Arg Lys and light heavy Arg Check l incorporation Expand cells for 6 doublings Optional Perform cell treatment NA Mix cells or medium from the two populations Prepare cell lysate and process lysates SDS PAGE Excise gel bands and perform In gel trypsin digestion Analyze tryptic peptides by MS Intensity m z L L L b EL Continued on next page Experimental Overview Continued Experimental Outline The experimental outline and general experimental timelines for using the SILAC kit are shown below See next page for the experimental workflow Step Action Page no Experimental Timeline Initiate your ES cell line of interest for growth Prepare mouse embryonic fibroblasts 11 39 Varies
37. ble Isotopes in Proteins for Accurate and Efficient Protein Identification Anal Chem 72 1134 1143 Coligan J E Dunn B M Ploegh H L Speicher D W and Wingfield P T 1998 Current Protocols in Protein Science Current Protocols Chanda V B Ed John Wiley and Sons Inc New York Goldsborough M Tilkins M L Price P Lobo Alfonso J Morrison J Stevens M Meneses J Pederson R Koller B and Latour A 1998 Serum Free Culture of Murine Embryonic Stem ES Cells Focus 20 8 12 Helmann U Wernstedt C Gonez J and Heldin C 1995 Improvement of an In Gel Digestion Procedure for the Micropreparation of Internal Protein Fragments for Amino Acid Sequencing Anal Biochem 224 451 455 Liang X Hajivandi M Veach D Wisniewski D Clarkson B Resh M D and Pope M R 2006a Quantification of Change in Phosphorylation of BCR ABL Kinase and its Substrates in Response to Imatinib Treatment in Human Chronic Myelogenous Leukemia Cells Proteomics 6 4554 4564 Liang X Zhao J Hajivandi M Wu R Tao J Amshey J W and Pope M R 2006b Quantification of Membrane and Membrane Bound Proteins in Normal and Malignant Breast Cancer Cells Isolated from the Same Patient with Primary Breast Carcinoma Journal of Proteome Research 5 2632 2641 Oda Y Huang K Cross F R Cowburn D and Chait B T 1999 Accurate Quantitation of Protein Expression and Site Specific Phosphoryla
38. cility such as the University of California San Francisco UCSF Stem Cell Facility follow the recommended protocols described by the core facility For details visit UCSF web site at http www escells ucsf edu researchers protocols asp For more technical resources on ES cells visit www invitrogen com stemcell If you have an optimized cell culture protocol for your ES cell line use the optimized protocol Follow the general guidelines below to grow and maintain ES cells e All solutions and equipment that come in contact with the cells must be sterile Always use proper sterile technique and work in a laminar flow hood e Before starting the labeling experiments be sure to have your ES cell line of interest established and have some frozen stocks on hand e Always use log phase cultures with gt 90 cell viability Determine cell viability using the trypan blue dye exclusion method e Culture ES cells in tissue culture plates such as 6 24 48 or 96 well plates rather than tissue culture flasks as the cells are difficult to scrape from flasks e Remove any differentiated cells from the plate prior to passaging the cells e Handle ES cells as potentially biohazardous material under the appropriate Biosafety Level as required by your institution e The MEM Non Essential Amino Acids Solution 10 mM 100X is irritating to eyes respiratory system and skin In case of contact with eyes rinse immediately with plent
39. ct for purposes other than research contact Licensing Department Invitrogen Corp 1600 Faraday Ave Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com References Amanchy R Kalume D E and Pandey A 2005 Stable Isotope Labeling with Amino Acids in Cell Culture SILAC for Studying Dynamics of Protein Abundance and Posttranslational Modifications Science s STKE 267 p12 Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Bae W and Chen X 2004 Proteomic Study for the Cellular Responses to Cd2 in Schizosaccharomyces pombe through Amino Acid Coded MassTagging and Liquid Chromatography Tandem Mass Spectrometry Mol Cell Proteomics 3 596 607 Beynon R J and Pratt J M 2005 Metabolic Labeling of Proteins for Proteomics Mol Cell Proteomics 4 857 872 Bodnar M S Meneses J J Rodriguez R T and Firpo M T 2004 Propagation and Maintenance of Undifferentiated Human Embryonic Stem Cells Stem Cells and Development 13 243 253 Chambers J L Christoph G G Krieger M Kay L and Sroud R M 1974 Silver Ion Inhibition of Serine Proteases Biochemical and Biophysical Research Communications 59 70 74 Chen X Smith L M and Bradbury M E 2000 Site Specific Mass Tagging with Sta
40. d inconsistent staining for pluripotency marker SSEA 4 lower panel A Undifferentiated hES cells B Differentiated hES cells Isotopic Labeling in Cell Culture Introduction Isotopic Labeling Labeling with Isotopically Labeled Amino Acid Instructions for performing cell labeling are described in this section At this point you should have initiated your ES cell line of interest for growth and prepared any frozen stocks if needed Metabolic labeling with stable isotope is performed using the SILAC technology To obtain complete incorporation of the isotope labeled amino acid into the proteins you need to adapt the cells to the medium containing the labeled amino acid Complete incorporation is usually achieved within 6 doublings of cells in this medium The SILAC Stem Cells Kit is supplied with U C L Lysine HCI MW 152 1259 which is a stable isotope of C L Lysine MW 146 1055 The heavy Lys is 6 daltons heavier than normal Lys For most of your experiments performing single labeling with Lys is sufficient to determine the relative expression of proteins Trypsin is the most widely used enzyme to generate peptides for MS analysis Trypsin cleaves proteins at the C terminus of arginine and lysine residues Cells labeled with heavy Lys and digested with trypsin yield peptides isotopically labeled with Lys When these labeled peptides with C terminal Lys are mixed with nonlabeled peptides and MS analys
41. daltons for peptides labeled with heavy Lys and U Ce 5N4 Arg respectively or 6 daltons for peptides labeled with heavy Lys and U Ce Arg when compared to peptides labeled with normal light Lys and Arg SDS PAGE Analysis Light Heavy r Samples were lysed and analyzed by SDS PAGE using NuPAGE Novex 4 12 Bis Tris Gel as described on the previous page and stained with a Coomassie stain Protein 1 bands 1 2 and 3 were excised from each side by side lane s 2 and subjected to in gel trypsin digestion and MS analysis see below u E MS Analysis MALDI TOF MS analysis was performed on samples using the Voyager DE STR MALDI TOF MS instrument A Lys containing peptide B Arg containing peptide 190 West L 1050 1p0 los 3556 1960 9 2 T 2 g44 1950 1956 1963 1969 1976 4328 i335 1342 i349 1356 1863 Xe D 100 100 1980 1 T 4 1347 9 2186 1969 1976 328 1335 1342 1349 1356 1963 Mass m z 1956 1963 Mass miz L Light Lys or Arg H U Cg4 Lys or U C 5N Arg 944 1950 22 Processing Samples Introduction Note Applications After performing cell labeling harvest the cells or media and process the samples for MS analysis Sample processing guidelines and workflows are described below Depending on the application that you wish to perform choose the appropriate workflow and guidelines e This section describes general workflows and guidelines
42. e supernatant and discard the nuclear pellet 6 Centrifuge the supernatant at 100 000 x g for 1 hour at 4 C to obtain the membrane pellet Carefully remove the supernatant and save the supernatant if you are interested in analysis of cytosolic proteins Continued on next page 43 Buffer Recipes Continued Phosphoprotein Lysis Buffer 44 The Phoshoprotein Lysis buffer contains strong detergents such as SDS for cell lysis and is mainly used for analysis of cytosolic and membrane associated proteins Liang et al 2006a This buffer is compatible with downstream applications such as SDS PAGE and immunoprecipitation Do not use this buffer if you wish to precipitate protein complexes as the buffer includes SDS 50 mM Tris HCI pH 8 0 1 Triton X 100 0 1 Sodium dodecyl sulfate SDS 0 5 Sodium deoxycholate 0 5 M NaCl 1 mM Sodium vanadate 10 mM Sodium fluoride Protease inhibitors 0 5 mM AEBSF 0 15 uM aprotonin and 1 uM leupeptin Store buffer at 4 C 1 Resuspend the ES cell pellet in 8 10 ml Phosphoprotein Lysis Buffer 2 Mix well by pipetting up and down 3 Centrifuge at 100 000 x g for 20 minutes at 4 C 4 Collect the supernatant lysate which contains the cytosolic and membrane associated proteins Save the pellet at 80 C if you are interested in analysis of membrane proteins General Methods for Protein Analysis Introduction Experimental Outline Note Important Materials Needed TM
43. ectly replaces FBS fetal bovine serum in existing protocols The performance of Knockout Serum Replacement is enhanced when used with Knockout D MEM Do not heat inactivate the Knockout Serum Replacement Do not use regular FBS to perform SILAC labeling experiments Trace amounts of amino acids present in regular FBS interfere with the incorporation of labeled amino acid and produce erroneous results TM GlutaMAX I Supplement L alanyl L glutamine is a dipeptide substitute for L glutamine and is supplied as a 200 mM 100X liquid stock in 0 85 NaCl GlutaMAX I can be used as a direct substitute for L glutamine at equimolar concentrations in cell cultures with minimal or no adaptation GlutaMAX I improves growth efficiency and performance of mammalian cell culture systems and eliminates problems associated with the spontaneous breakdown of L glutamine during incubation GlutaMAX Iis highly soluble in aqueous solution and is heat stable Continued on next page Description of Kit Components Continued MEM Non Essential MEM Non Essential Amino Acids Solution 10 mM 100X is prepared in Amino Acids Solution SILAC Amino Acids Note distilled water and the non essential amino acids in this solution are 100X the concentration in a MEM alpha Medium For detailed formulation visit www invitrogen com SILAC Amino Acids are highly pure cell culture grade amino acids used for supplementing the basal media to
44. efault Open Search Template Save Search Template As All Searches MASCOT Server RNDGPS3 Taxonomy All entries Report Top 200 Hits Database NCBI Enzyme Trypsin Max Missed Cleavages Fixed Modifications N term iTRAQ Variable Modifications g e AB old ICATdO C 3 B old ICATd8 C s vy Acetyl K Arg light Acetyl N term Arg heavy Protein Mass kDa MS MS Fragment Tol 0 15 Da v Precursor Tolerance 75 ppm X Monoisotopic Average Peptide Charges 1 Species Info Required Filtering Maximum Peptide Rank 10 Minimum lon Score C I Peptide 0 IV Perform Quantitation ICAT C iTRAQ ICAT Delta Mass FragmentMass Selected Reference ICAT Pair Tolerance Fragment Tolerance Da Save Settings Cancel An example of results using the GPS Explorer software is shown on the next page Continued on next page 33 Protein Identification and Quantitation continued Example of GPS An example of quantitation result based on a pair of light and heavy Lys Explorer peptides obtained after analysis using GPS Explorer software is shown below Analysis Results To view the quantitation results review the data in the column Avg ICAT Ratio H L indicated with a circle in the figure below For down regulated proteins the ratio will be less than 1 and for up regulated proteins the ratio will be greater than 1 See next page for de
45. egular and conditions SILAC ES Cell 1 Media Remove the MEF medium and wash cells with PBS Perform a final wash with SILAC KnockOut D MEM without any supplements or amino acids Add 2 ml trypsin solution to the flask and incubate for 5 minutes Add 2 ml MEF medium and transfer the cells to a sterile 15 ml centrifuge tube and mix well Perform a cell count using Trypan Blue Irradiate MEF cells with 40 Gy or treat MEF cells with Mitomycin C 10 ug ml for 2 hours to arrest the growth without killing the cells Centrifuge the cells at 800 x g for 5 minutes and resuspend the cells in MEF medium Plate the cells in two 1 25 flasks or 6 well plates at a cell density of 55 000 cells cm The next morning wash two flasks of MEF cells with PBS three times Add 1 3 ml well for a 6 well plate or 5 ml per T25 flask of the following medium e Light or heavy SILAC ES cell media containing 4 ng ml human bFGF or 1000 units ml LIF prepared as described on page 13 to the MEF cells e Regular ES Cell Medium containing 4 ng ml human bFGF or 1000 units ml LIF prepared as described on page 14 to the MEF cells Incubate the MEF cells in Regular or SILAC light or heavy ES cell media in a 37 C incubator containing a humidified atmosphere of 5 CO in air overnight After overnight incubation collect the respective MEF conditioned ES cell media from feeder flask or plate Supplement the conditioned medium with additional 4
46. ells or 1000 units ml LIF mouse ES cells Optional Phenol Red solution Do not add any Penicillin Streptomycin to human ES cells as human ES cells are sensitive to antibiotics 1 Resuspend 100 mg L Lysine HCl and 100 mg U C L Lysine Lys each in 1 ml basal unsupplemented SILAC KnockOut D MEM Mix well until completely dissolved 2 Resuspend 100 mg L Arginine from each vial 2 vials are supplied in the kit in 1 ml basal unsupplemented SILAC KnockOut D MEM Mix well until completely dissolved Note If you are using double labeled arginine available separately from Invitrogen see page vii resuspend 100 mg U Cz 5N4 L Arginine Arg or 100 mg U C L Arginine Arg in 1 ml basal unsupplemented SILAC KnockOut D MEM supplied with the kit Mix well until completely dissolved 3 Transfer 80 ml SILAC KnockOut D MEM to two separate 100 ml sterile filter unit with 0 22 um membrane includes membrane filter funnel and receiver bottle Add 20 ml KnockOut Serum Replacement to each filter unit To one filter device from Step 4 add 0 1 ml L Lysine HCl 100 mg ml from Step 1 and 0 1 ml L Arginine 100 mg ml from Step 2 to prepare light SILAC ES Cell medium supplemented with light normal lysine and arginine Mark the bottle appropriately Store the remaining lysine and arginine solution at 20 C 6 To the second filter unit from Step 4 add 0 1 ml Lys 100 mg ml from Step 1 and 0 1 ml
47. ental workflow for using the SILAC Stem Cells Kit Culture ES cells using MEP eS ha Prepare mouse conditioned embryonic SLAC mdm fibroblasts MEF Prepare SILAC Media with Grow one cell GrOW Oten supplements d cell population amino acids r population in I in heavy light medium medium Y Y No Expand cells for 6 Expand cells for 6 No doublings doublings y Apply treatment to either cell population Harvest cells or medium Mix cells or medium from both populations Prepare samples Run the gel and using standard stain the gel protein peptide separation methods Perform in gel trypsin Core facility No digestion performs in gel Transfer trypsin digestion MS Yes samples to core analysis protein ID facility and quantitation Transfer Core facility performs MS tryptic k gt analysis protein peptides to core facility ID and quantitation Continued on next page Methods Before Starting Important Efficiency of Incorporation MS Core Facility TM e Review the information in this section prior to starting your SILAC experiments You need to perform certain experiments and need to purchase some reagents before proceeding with isotope labeling experiments
48. fer containing the tyrosine phosphatase inhibitor to prevent any loss of phosphorylation page 43 Do not concentrate the peptides to dryness after trypsin solution If the peptides are concentrated to dryness the peptides are difficult to resuspend resulting in loss of peptides To enable proper data analysis and identification of phosphorylated proteins always perform enrichment of phosphoproteins prior to analysis Perform labeling for at least 6 doublings to ensure complete incorporation of the label Be sure to use log phase cells with gt 90 viability The SILAC Kit is designed to provide gt 90 incorporation of labeled amino acids page 2 Review the solutions below to prevent introduction of exogenous light amino acids at specific steps e Dilute Matrigel with SILAC KnockOut D MEM without Lys Arg Gln as described on page 42 Do not use SILAC KnockOut D MEM supplemented with Lys Arg Gln Prepare Collagenase IV solution in SILAC KnockOut D MEM without Lys Arg Gln as described on page 19 Wash the MEF cells thoroughly with PBS to remove any traces of FBS that may contain amino acids Perform a final wash with SILAC KnockOut D MEM without Lys Arg Gln prior to conditioning the medium as described on page 41 TM Ensure the growth factors used for ES cell culture do not contain any exogenous amino acids Appendix Preparing Mouse Embryonic Fibroblasts MEF Introduct
49. ht normal medium and heavy isotope labeled medium in a 1 1 ratio based on the cell number Centrifuge the cells at 500 x g for 5 minutes at 4 C to remove PBS Resuspend the cell pellet in a hypotonic buffer to isolate plasma membrane or in a non ionic detergent containing buffer to isolate cytosolic and membrane associated proteins Be sure to use appropriate protease inhibitors and phosphatase inhibitors in the buffer See page 43 for buffer recipes and protocols to prepare cell lysates Mix well by pipetting up and down Centrifuge the lysate as follows e For cytosolic and membrane associated proteins centrifuge at 100 000 x g for 20 minutes at 4 C Collect the supernatant containing cytosolic and membrane associated proteins Save the pellet at 80 C if you are interested in analysis of membrane proteins e For membrane proteins centrifuge at 100 000 x g for 60 minutes at 4 C Collect the pellet containing membrane proteins Save the supernatant at 80 C if you are interested in analysis of cytosolic proteins 9 Separate the proteins by SDS PAGE using NuPAGE Novex precast gels page 47 Transfer stained gel to the proteomics core facility or proceed to the next step 10 Perform in gel trypsin digestion page 49 11 Proceed directly to MS analysis after trypsin digestion page 27 Submit your tryptic peptides to the proteomics core facility for analysis 12 After data analysis perform crossover experiments to verif
50. ides Shevchenko et al 1996 To avoid keratin contamination perform the in gel digestion in a laminar flow hood 1 Rinse the stained gel in water for 10 minutes to remove any particulate material 2 Excise the desired gel band from the stained gel Mince the excised gel piece into smaller pieces 1 mm x 1 mm Transfer the gel pieces to a clean microcentrifuge tube 3 Add 500 ul 50 acetonitrile 25 mM ammonium bicarbonate pH 8 0 Incubate at room temperature for 15 30 minutes for destaining the gel pieces Vortex for 10 seconds and briefly centrifuge at maximum speed for 10 seconds Discard the supernatant carefully without removing gel pieces 4 RepeatStep 3 until the gel pieces are sufficiently destained 5 Add 200 ul 100 acetonitrile to dehydrate the gel pieces Incubate for 5 10 minutes at room temperature Vortex for 10 seconds and briefly centrifuge at maximum speed for 10 seconds Discard the supernatant carefully without removing any gel pieces 7 Drythe gel pieces in a centrifugal vacuum concentrator e g Thermo Savant SpeedVac centrifuge 8 Add enough cold trypsin solution 10 ng pl in 25 mM ammonium bicarbonate pH 8 0 to cover the gel pieces 9 Incubate on ice for atleast one hour to allow the trypsin solution to penetrate the gel pieces The cold temperature helps to prevent autolysis of the trypsin 10 Incubate overnight at 37 C 11 Add 50 ul 5 formic acid FA and incubate for 30 minutes at
51. ight and heavy Lys and a pair of light and heavy Arg An example of the Mascot search result is shown on the next page Continued on next page 31 Protein Identification and Quantitation Continued Example of Mascot Search Result Protein Quantitation GPS Explorer 32 The Mascot search result shows identities of proteins and the output shows peptides labeled with light or heavy Lys and or Arg as shown below Peptide Summary Report Switch to Protein Summary Report To create a bookmark for this report right click this link Peptide Summary Report SampleSetID 343 AnalysisID 818 Path Gqui Membrane msilacexp 3bqand12jrk lysargmsms Select All Select None Search Selected Error tolerant Archive Report 1 gi 5453832 Mass 111266 Total score 729 Peptides matched 37 oxygen regulated protein precursor oxygen regulated protein 150kD Homo sapiens Check to include this hit in error tolerant search or archive report Query bserved Mr expt Mr calc Delta Miss Score Rank Peptide M 23 1005 51 1004 50 1004 54 0 05 0 36 1 YFQHLLGK Moa 1011 54 1010 53 1010 54 0 01 0 48 1 YEQHLLGK Lys heavy M a8 1011 55 1010 54 1010 54 0 00 0 35 1 YEQHLLGK Lys heavy M a9 1012 54 1011 53 1011 55 0 01 0 41 1 EVQYLLNK Lys heavy M so 1012 56 1011 55 1011 55 0 00 0 48 1 EVQYLLNK Lys heavy 131 1041 49 1040 48 1040 48 0 00 1 15 7 KYPDYESK 2 Lys_heavy M 161 1047 53 1046 53 1046 55 0 03 0 32 1 LAGLFHEQR Iv i62 10
52. ion Important Note Experimental Outline Materials Needed TM The SILAC Stem Cells kit is designed for culturing human ES cells maintained in a defined media that is conditioned using MEF cells Instructions to prepare MEF from mouse embryos and prepare MEF conditioned SILAC media are described in this section The MEF preparation protocol described in this section is suitable to obtain MEF cells for conditioning SILAC media General guidelines and a brief protocol for preparing MEF is described below If you have access to a stem cell core facility such as the University of California San Francisco UCSF Stem Cell Facility follow the recommended protocols described by the core facility For details visit UCSF web site at http www escells ucsf edu researchers protocols asp For more technical resources on ES cells visit www invitrogen com stemcell If you have an optimized MEF preparation protocol use the optimized protocol Follow the general recommendations and guidelines of the animal care committee for your local institution during animal dissection and disposal Use approved methods for animal handling and dissection Contact your local animal care committee for more information Dissect a 13 day pregnant mouse and remove the embryo Isolate MEF cells from the embryo using trypsin Triturate the MEF cells and culture the MEF cells in MEF medium Subculture the cells and freeze some MEF cells for
53. ioned SILAC medium containing heavy lysine and heavy arginine prepared as described on pages 13 and 41 Incubate the dishes in a 37 C incubator containing a humidified atmosphere of 5 CO in air The day after seeding undifferentiated ES cells are seen as small colonies while single cells begin to differentiate Grow the cells separately and change the medium every day using the appropriate MEF conditioned SILAC light or heavy ES Cell medium Passage the cells at a ratio of 1 3 to 1 6 when cells reach 70 80 confluency 4 7 days Note Cells grow at a similar rate in each media Expand each cell population for at least six doubling times usually is about 2 weeks to achieve gt 90 incorporation of labeled amino acid into the proteins After six doublings harvest a small aliquot of cells 10 cells from each cell population to determine the efficiency of incorporation Store the cell pellet at 80 C until use See next page for details on sample processing At the end of six doublings you will have 6 4 x 106 cells for each cell population Based on the type of analysis that you wish to perform the approximate number of cells needed for each analysis from each cell population are listed below e 60 x 10 or cells from 2 x 100 mm plates for identifying secreted growth factors e 30 x 10 or cells from 1 x 100 mm plate for studying differential protein expression e 107 108 or cells from 2 to 3 x 100 mm plate fo
54. is is performed peptides are detected as peak pairs that are precisely 6 0204 Da apart Using labeling with Lys only you detect peak pairs only for the subset of peptides with C terminal Lys residues while detecting the peptides with C terminal Arg residues as superimposed singlet peaks If you need maximal sequence coverage or need to monitor all possible phosphorylation sites we recommend performing a double labeling experiment wherein the proteins are labeled with U C L Lysine HCl and U C N4 L Arginine MW 184 1241 Arg containing peptides ionize better in MALDI MS than Lys containing peptides resulting in better sensitivity and sequence coverage Using double labeling increases the number of informative peptides making the method more sensitive U C N 4 L Arginine Arg is available separately from Invitrogen page vii and is a stable isotope of C amp N4 L Arginine MW 174 1117 After trypsin digestion and MS analysis you will observe peak pairs that are separated by 6 0204 Da for Lys and Lys pairs and 10 0124 Da Arg and Arg pairs Continued on next page 17 Isotopic Labeling in Cell Culture continued Experimental 1 Harvest ES cells and initiate two cultures Grow one culture in the MEF Outline conditioned SILAC light normal supplemented medium and the other culture in MEF conditioned SILAC heavy isotope labeled supplemented medium 2 Grow the two ES cell populations for at least s
55. its were developed using the 4700 Proteomics Analyzer MALDI TOF TOF equipped with GPS Explorer software Applied Biosystems and Q Tof Platform Waters that allowed protein identification and quantitation after labeling On page 30 we describe the procedures to set up SILAC quantitation with data collected on ABI MDS Sciex Family of MALDI TOF TOF equipped with GPS Explorer Software as an example TM SILAC quantitation is now supported by software platforms running each of the major proteomic tandem MS instruments See the table below for a list of tandem MS instrument and the corresponding software packages that support SILAC data analysis Instrument Manufacturer ABI MDS Sciex Family of MALDI TOF TOF Applied Biosystems including the 4700 and 4800 Proteomics Analyzer platforms equipped with GPS Explorer Software versions 3 1 and higher QSTAR and Q TRAP LC MS MS systems with Applied Biosystems ProteinPilot software Q Tof Platform with ProteinLynx Global SERVER Waters software version 2 2 5 and higher LCQ and LTQ Traps as well as the Orbitrap and Thermo Fisher LTO FT may be purchased with or supplemented by Scientific add on of BioWorks software version 3 3 and higher Because specific software details may vary among instrument manufacturers we recommend that you consult with your proteomics MS core facility If you have other MS instruments you can perform semi aut
56. ix doublings to allow complete incorporation of the labeled amino acid 3 Perform the cell treatment see below if appropriate Treatment of Cells You may induce cell differentiation using any growth factor or stimulant Since the SILAC labeling experiments are performed in cell culture various types of cell treatments can be performed to compare the effect of the treatment on protein expression The time for the treatment is highly variable from 5 30 minutes to several days depending on the treatment Materials Needed e Human or mouse ES cells see page 15 for the number of cells needed for labeling e MEF conditioned SILAC heavy and light ES Cell medium page 41 e Collagenase IV page vii e Optional growth factors if needed for your cells e Appropriate tissue culture dishes and flasks e Matrigel coated plates page 42 e 37 C incubator with a humidified atmosphere of 5 CO in air e Sterile centrifuge tubes e Reagents to determine viable and total cell counts page vii e Appropriate reagents for cell treatment if applicable For determining the efficiency of incorporation you also need e NuPAGE LDS Sample Buffer 4X e NuPAGE Sample Reducing Agent 10X e NuPAGE Novex Bis Tris Gel e NuPAGE MES MOPS SDS Running Buffer 20X Continued on next page 18 Isotopic Labeling in Cell Culture Continued Before performing the isotopic labeling experiments be sure Note You have the required numbe
57. l amino acid into the sample Using SILAC technology for quantitative proteomics in embryonic stem cells offers the following advantages e Simple easy to use labeling protocol designed for cell biologists and protein biochemists and performed using standard laboratory equipment e Produces gt 90 labeling efficiency as compared to other labeling methods currently available e Allows specific sequence labeling of peptides since isotope labeled amino acid medium is used instead of isotopic nuclei labeled medium e Generates uniformly labeled proteins to analyze several peptides for accurate results and increased sequence coverage e Eliminates quantitation error due to unequal sample preparation and increases reproducibility as the two cell populations are mixed after treatment and treated as a single sample in all subsequent steps e Provides flexibility in the choice of amino acid used for labeling and the types of treatment that can be applied to cells Continued on next page Overview Continued Applications System Overview Important Purpose of the Manual SILAC technology can be used to e Identify secreted growth factors e Quantitatively analyze differential protein expression in the presence of growth factors or a stimulus e Study signal transduction pathways i e changes in phosphorylation lipidation relocation ubiquitination upon stimulation To perform quantitative analysis of protein expression using SIL
58. ling experiments and then coordinate with the proteomics core facility for sample processing and MS analysis Based on your expertise with certain protocols and the options provided by the core facility you can transfer the samples to the core facility for MS analysis at various points as indicated in the protocols As each core facility has specific requirements for sample preparation and handling it is important that you consult with your core facility about the sample requirements prior to preparing the samples You also need to work closely with the core facility to schedule time for the MS analysis when your samples are ready Recommended Core Facilities for SILAC If you do not have access to a core facility or the core facility is not equipped to perform MS analysis for SILAC contact Technical Support page 50 for a list of recommended core facilities We have identified and qualified some core facilities for performing MS analysis protein identification and quantitation for SILAC Continued on next page Before Starting Continued Note MS Instruments Antibodies 10 If you are an experienced user of MS have access to various MS instruments and are able to perform MALDI MS or LC MS analysis you may chose to perform the MS analysis yourself without working with a core facility SILAC experimental data can be analyzed using MALDI TOF MS analysis for simple samples or using MS MS analysis for complex samples SILAC K
59. lows The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for activities in the Excluded Fields The Excluded Fields include a the implantation or production for implantation of primate Pluripotent Stem Cells pPSC s or cells or materials derived from pPSCs into an animal by or in collaboration with a for profit entity b the manufacture of therapeutic products diagnostic products or components of either c the manufacture of a product for sale or for use in commercial services or the use of a product in commercial services and or d the production of materials for use in human clinical trials Through its use of this product the purchaser names Geron Corporation as a third party beneficiary of this license for the sole purpose of enforcing such license Parties wishing to use this product for applications within the Excluded Fields will require and must request a direct license from Geron Such a license request should be sent to Geron Corporation Attn Legal Dept 230 Constitution Drive Menlo Park California 94025 If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For additional information on purchasing a license to this produ
60. m e Human ES cells human bFGF to a final concentration of 4 8 ng ml e Mouse ES cells murine LIF to a final concentration of 1000 units ml 7 Store the medium at 2 to 8 C protected from light until use The medium is stable for 2 weeks when properly stored avoid introducing any contamination into the medium Continued on next page Preparing Cells and Media Continued Culturing ES Cells Cells for Labeling After receiving the ES cells follow the manufacturer s recommendations to thaw and subculture the human or mouse ES cells For more technical resources on ES cells visit www invitrogen com stemcell See the next page for examples of ES cell images when cultured on MEF conditioned KnockOut D MEM Use the MEF conditioned Regular ES Cell Medium prepared as described page 41 to culture the ES cells Do not use the SILAC ES Cell Medium for routine ES cell culture The protocols in this manual are designed for culturing ES cells on Matrigel coated plates See page 42 for preparing Matrigel coated plates If you have an optimized ES cell culture protocol using gelatin coated plates use the protocol optimized for your ES cell line The SILAC Stem Cell Kit is designed for ES cell culture using MEF conditioned medium During SILAC labeling experiments the ES cells are maintained on MEF conditioned SILAC heavy and light ES Cell Medium See page 41 to prepare MEF and MEF conditioned SILAC heavy and light ES Cell
61. ncy of labeling for any analysis type Note Since ES cells are usually passaged as colonies varying from 50 500 cells colony approximate cells 100 mm plate were used for cell number calculations Continued on next page 15 Preparing Cells and Media Continued Images of ES Cells 16 Images of undifferentiated and differentiated human ES cells grown in MEF conditioned KnockOut D MEM are shown below as examples For more technical resources on ES cells including ES cell images visit www invitrogen com stemcell Colonies of human ES cell line were plated onto a synthetic matrix surface in MEF conditioned KnockOut D MEM containing Knockout Serum Replacement and 4 ng ml bFGF After 3 days of cultivation the colonies were fixed in 4 formaldehyde rinsed with D PBS immunostained with SSEA 4 stage specific embryonic antigen 4 antibody fluorescently labeled with goat anti mouse IgG Alexa Fluor 488 and stained with nuclear dye DAPI Colonies were visualized using a fluorescent microscope with 10x objective Results The images for undifferentiated cells panel A illustrate good compact morphology of individual cells uniform colony clump with a well defined edge upper panel and shows good staining for a known pluripotent state marker SSEA 4 lower panel The images for differentiated cells panel B illustrate breaking of the human ES colony as cells begin to migrate distorting colony edge upper panel an
62. ng ml of human bFGF or 1000 units ml LIF before using the conditioned medium to expand human ES cells Use the irradiated MEF for up to 6 7 days and collect the conditioned medium once every day 41 Preparing Matrigel Coated Plates Introduction amp 1 y MOMENO 7 8 v e P Materials Needed Protocol 42 TM The ES cells are usually grown on Matrigel or gelatin coated plates Matrigel is a basement membrane matrix that provides support for growth Instructions to prepare Matrigel coated plates for growth of ES cells are described in this section If you have established protocols for growing ES cells using gelatin coated plates use the established protocols TM e Maintain the Matrigel at 4 C to prevent the gel from solidifying during pipetting e Keep the tissue culture plates pipettes and medium cold e Matrigel Growth Factor Reduced GFR Basement Membrane Matrix Phenol Red free Becton Dickinson catalog no 356231 e 6 well tissue culture plate and sterile pipettes e SILAC KnockOut D MEM without Arg Lys Gln phenol red supplied with the kit chilled at 4 C 1 Thaw aliquots of Matrigel at 4 C until the gel liquifies 2 Dilute Matrigel 30 fold with chilled SILAC KnockOut D MEM without Arg Lys Gln phenol red 3 Add 1 ml diluted Matrigel solution to coat a 6 well plate Incubate for 1 2 hours at room temperature Remove any non polymerized Matrigel s
63. nute at 10 000 x g and load supernatant onto a NuPAGE Novex Bis Tris Gel and analyze the protein immune complexes using SDS PAGE below SDS PAGE The following procedure uses NuPAGE Novex Bis Tris Gels with the XCell Analysis SureLock Mini Cell If you are using any other electrophoresis system refer to the manufacturer s recommendations 1 Assemble the gel cassette Buffer Core sandwich as described in the XCell SureLock Mini Cell manual download the manual from www invitrogen com If you are using only one gel use the Buffer Dam to replace the second gel cassette 2 Fillthe Lower Buffer Chamber and Upper Buffer Chamber with the recommended volume of 1X NuPAGE MES or MOPS SDS Running Buffer Add 0 5 ml of NuPAGE Antioxidant to the Upper Buffer Chamber 3 Load the processed samples and load protein molecular weight standards in a different well 4 Place the XCell SureLock Mini Cell lid on the Buffer Core With the power on the power supply turned off connect the electrode cords to the power supply 5 Perform SDS PAGE at 200 V for 40 50 minutes for NuPAGE Novex Bis Tris Gel 6 Atthe end of electrophoresis turn off the power and disassemble the gel cassette Buffer Core sandwich assembly as described in the XCell TM SureLock Mini Cell manual 7 Proceed to gel staining next page Continued on next page 47 General Methods for Protein Analysis Continued Staining the Gel Note p b D 4
64. oethanol 1 000X Liquid 55 mM Gel ice 2 8 C in Dulbecco s Phosphate Buffered Saline D PBS Media Kit The kit components for SILAC Stem Cells Protein ID and Quantitation Media Components Kit with U C L Lysine Lys and KnockOut D MEM are listed below TM Store all components at 2 8 C except KnockOut Serum Replacement and GlutaMAX I Supplement which are stored at 5 to 20 C Component Amount SILAC KnockOut D MEM 2 x 1000 ml KnockOut Serum Replacement 500 ml GlutaMAX I Supplement 100 ml MEM Non Essential Amino Acids 100 ml Solution 10 mM 100X liquid 2 mercaptoethanol 1 000X 50 ml SILAC Phenol Red Solution 10 g L 5ml SILAC L Lysine HCl 100 mg SILAC L Arginine 2 x 100 mg SILAC U C4 L Lysine HCl Lys 100 mg vi Accessory Products SILAC Kits The table below lists additional SILAC Kits available separately For more information visit www invitrogen com or call Technical Support page 50 Product Quantity Catalog no SILAC U 8C N L Arginine Arg 100 mg MS10009 SILAC U 8C L Arginine Arg 100 mg MS10011 SILAC Protein Identification and Quantitation Media Kit with U Cg L Lysine Lys and D MEM Flex 1 kit MS10030 with U C L Lysine Lys and RPMI Flex 1 kit MS10031 with U P Cg L Lysine Lys and IMDM Flex 1 kit MS10032 with U C L Lysine Lys and Advanced D MEM F 12 Flex 1 kit MS10033
65. of 1000 units ml 11 Store the medium at 2 to 8 C protected from light until use The medium is stable for 2 weeks when properly stored avoid introducing any contamination into the medium Prepare the Regular ES Cell Medium 100 ml as described below Adjust the reagent volumes accordingly to prepare gt 100 ml medium You need to purchase the media components separately from Invitrogen page vii To prepare SILAC ES Cell Medium see page 13 Perform all steps in a tissue culture hood under sterile conditions KnockOut D MEM 20 KnockOut Serum Replacement 2 mM L GlutaMAX I Supplement 0 1 mM MEM Non Essential Amino Acids Solution 1X 55 uM 2 mercaptoethanol 4 8 ng ml human bFGF human ES cells or 1000units ml LIF mouse ES cells Do not add any Penicillin Streptomycin to human ES cells 1 Transfer 80 ml KnockOut D MEM to a 100 ml sterile filter unit with 0 22 um membrane includes membrane filter funnel and receiver bottle Add 20 ml KnockOut Serum Replacement to the filter unit To the filter unit add 1 ml 100X L GlutaMAX I 1 ml 100X MEM Non Essential Amino Acids Solution 0 1 ml Phenol Red Solution and 0 1 ml 1 000X 2 mercaptoethanol 4 Optional You may supplement the medium with additional growth factors or cytokines if needed for your specific cell line Apply vacuum to the filter unit to filter sterilize each medium To culture ES cells add the following growth factors to each 100 ml mediu
66. olution from the plate before use TM 4 Use the Matrigel coated plates immediately for culture of ES cells or wrap the plates with a plastic wrap and store the plates at 4 C The Matrigel coated plates are stable for 1 week at 4 C Buffer Recipes Introduction Membrane Lysis Buffer Recipes for recommended buffers for lysis of cells for phosphoprotein analysis or to solubilize membrane proteins are described below If you have an optimized buffer recipe for your application that produces efficient lysis use the optimized buffer recipe The Membrane Lysis Buffer is a hypotonic lysis buffer and is used with 1 25 M sucrose solution for cell lysis Liang et al 2006b This buffer is compatible with downstream applications such as immunoprecipitation and SDS PAGE 10 mM Tris HCI pH 8 0 1 mM Magnesium chloride Protease inhibitors 0 5 mM AEBSF 0 15 uM aprotonin and 1 uM leupeptin Store buffer at 4 C Before using for lysis add 40 ul Benzonase Nuclease to 50 ml Membrane Lysis Buffer and store on ice until use 1 Resuspend the ES cell pellet in 1 6 ml Membrane Lysis Buffer Mix well by pipetting up and down Incubate on ice for 30 minutes Homogenize the lysate on ice using a Dounce homogenizer or equivalent for 30 strokes 4 Add 0 4 ml 1 25 M sucrose solution to the lysate and mix well by pipetting up and down 5 times 5 Centrifuge the lysate at 500 x g for 10 minutes at 4 C to remove nuclear fraction Remove th
67. omated analysis of SILAC raw data using the MS instrument for protein identification but you need to perform protein quantitation using manual calculations contact the instrument vendor A large variety of antibodies against various proteins is available from Invitrogen page vii Antibodies against specific epitope tags such as 6X His V5 Myc are also available from Invitrogen Visit our website at www invitrogen com for more information Preparing Cells and Media Introduction ES Cells Important General Guidelines To perform SILAC experiments you need a human or mouse ES cell line of choice General guidelines are included below for handling ES cells and preparing the media If you are performing ES cell culture for the first time refer to published protocols for more information Bodnar et al 2004 For more technical resources on stem cells visit www invitrogen com stemcell The SILAC labeling does not affect the growth or pluripotency of ES cells The ES cell line of choice must be able to grow in KnockOut D MEM supplemented with KnockOut Serum Replacement under the conditions used for labeling see page 20 for details If your ES cells require specific growth factors for growth you may add the growth factors to the medium but do not add any additional amino acids to the growth medium General guidelines for ES cell culture are described in this section If you have access to a stem cell core fa
68. performing a crossover experiment Briefly the crossover experiment involves repeating the experiment except that you perform cell treatment to the other cell population for example if you stimulated the heavy labeled cells then stimulate the light labeled cells to perform the crossover experiment Peptides derived from secreted proteins in a stimulus specific response appear more abundant from the heavy labeled medium in the first experiment while in crossover experiment the peptides appear more abundant from the light labeled medium Identified proteins that do not have a reciprocal response between the two experiments are more likely to be background proteins Continued on next page Processing Samples Continued Differential Protein Expression Analysis Note The general workflow for analyzing differential protein expression or identifying unique cell surface markers upon differentiation using labeled ES cells is described below For this application you need 30 x 10 cells or 1 x 100 mm plate of cells from each cell population After performing the labeling for six doubling times and performing the cell treatment if appropriate harvest cells from each ES cell population as below 1 Determine the viable and total cell count on an aliquot of cells using the trypan blue method Harvest the required number of ES cells from each population page 21 Resuspend each cell pellet in 1 ml chilled PBS Mix the cells grown in lig
69. pletely destained no yellow color is visible before trypsin digestion After staining the gel you may transfer the stained gel to the core facility to perform in gel trypsin digestion and MS analysis as described in this manual If you wish to stain the gel and perform in gel trypsin digestion follow the protocol described on the next page For more information on proteomics core facilities that offer MS analysis for SILAC see page 9 Follow these guidelines for trypsin digestion to obtain the best results e Always use sequencing proteomics grade trypsin for MS analysis page vii e Always prepare the trypsin digestion buffer 25 mM ammonium bicarbonate buffer pH 8 0 using ultra pure reagents and water e Avoid touching the gel with bare hands to prevent contamination from keratin e Be sure to use polypropylene microcentrifuge tubes and HPLC grade solvents to avoid any contamination from polymers Continued on next page General Methods for Protein Analysis Continued In gel Trypsin A general protocol for in gel trypsin digestion is provided below You may use Digestion any method of choice or a method recommended by your proteomics core facility For more information refer to published reference sources Coligan et al 1998 Helmann et al 1995 Note The digestion protocol given below is generally used for protein identification If you need more sequence coverage you may need to perform reduction and alkylation of pept
70. prepare complete media The SILAC Amino Acids include the normal light and isotope labeled heavy amino acids SILAC Light Amino Acids The SILAC Kits include L Lysine HCI and L Arginine as light unlabeled amino acids These amino acids are normal essential amino acids and do not contain any isotopic label Use the light amino acids to prepare the light unlabeled medium as directed in the protocol page 13 SILAC Heavy Amino Acid The SILAC Heavy Amino Acid includes the heavy isotope labeled amino acid U C L Lysine HCl MW 152 1259 The labeled Lys is a stable isotope of C4 L Lysine MW 146 1055 The Lys is 6 daltons heavier than the light L Lysine Use the heavy amino acid to prepare the heavy labeled medium as directed in the protocol page 13 If you need maximal sequence coverage or need to monitor all possible phosphorylation sites we recommend performing a double labeling experiment wherein the proteins are labeled with U C L Lysine and U C N 4 L Arginine or U C L Arginine See page 17 for details U 8Ce Na L Arginine and U C L Arginine available separately from Invitrogen page vii are stable isotopes of 7C Na L Arginine and C L Arginine respectively After trypsin digestion and MS analysis you will observe peak pairs that are separated by 10 Da for Arg and U C N4 L Arg pairs or 6 Da for Arg and U C L Arg pairs Arg containing peptides ionize
71. r of ES cells actively growing with gt 90 viability To keep some cells aside to measure the percentage of incorporation as directed in the protocol To prevent introduction of exogenous light amino acids at specific steps follow these guidelines e Dilute Matrigel with SILAC KnockOut D MEM without Lys Arg Gln as described on page 42 Do not use SILAC KnockOut D MEM supplemented with Lys Arg Gln e Prepare Collagenase IV solution in SILAC KnockOut D MEM without Lys Arg Gln as described on page 19 e Wash the MEF cells thoroughly with PBS to remove any traces of FBS that may contain amino acids Perform a final wash with SILAC KnockOut D MEM without Lys Arg Gln prior to conditioning the medium as described on page 41 e Ensure the growth factors used for ES cell culture do not contain any exogenous amino acids Harvesting ES Once you have established the ES cell line and cells have undergone at least Cells 6 doublings use the ES cells for SILAC labeling 1 Prepare 200 units ml Collagenase IV solution in SILAC KnockOut D MEM without adding any supplements deficient in Lys Arg Gln and phenol red Filter sterilize using 0 22 um filtration device Remove the medium and wash the cells once with PBS Add 1 ml 200 units ml Collagenase IV to each well of a 6 well plate for each cell population Use one 6 well plate for heavy and light labeling each Incubate at 37 C for 5 10
72. r studying signal transduction pathway Note You may freeze the remaining cells or continue to maintain or expand the two cell populations in the light or heavy medium if you wish to repeat the experiment Proceed to Performing the Cell Treatment next page if needed or Processing Samples page 23 Continued on next page Isotopic Labeling in Cell Culture continued Performing Cell Treatment Determining the Efficiency of Incorporation After verifying that the you obtain gt 90 incorporation efficiency see below perform the cell treatment as described below You may label the cells in light or heavy medium 1 Determine the viable and total cell count using the trypan blue exclusion method Save an aliquot of cells as control prior to starting the treatment To either cell population induce cell differentiation using growth factors or a stimulus Perform the treatment for the desired time usually 5 30 minutes to several days depending on the treatment At the end of the treatment proceed to Processing Samples page 23 To ensure gt 90 incorporation of the heavy amino acid into proteins analyze small aliquots of cells 10 labeled with light or heavy amino acids and determine the efficiency of incorporation 1 6 7 8 After six doublings harvest a small aliquot of cells 10 cells from each cell population as described in Step 7 previous page Lyse each cell pellet separately in 180 ul 1X
73. rcaptoethanol 100 mg L L Arginine 4 8 ng ml human bFGF for human 2 mM L GlutaMAX I Supplement ES cells or 1000 units ml LIF for 0 1 mM MEM Non Essential Amino Acids mouse ES cells 1X 55 pM 2 mercaptoethanol 4 8 ng ml human bFGF for human ES cells or 1000 units ml LIF for mouse ES cells Optional Phenol Red solution Thaw ES cells and maintain the ES cells in MEF conditioned Regular ES Cell Medium Harvest ES cells using Collagenase IV solution and resuspend the ES colonies in appropriate MEF conditioned SILAC Light or Heavy ES Cell Medium Transfer the ES cells as follows into 35 mm MatriGel coated plates to obtain a seeding density of 5 x 10 1 5 x 10 cells cm cell culture plate e Light Cell Population Transfer the ES cells in 4 ml MEF conditioned SILAC medium containing light lysine e Heavy Cell Population Transfer the ES cells in 4 ml MEF conditioned SILAC medium containing heavy lysine Incubate in a 37 C incubator containing a humidified atmosphere of 5 CO in air Grow the cells separately and change the medium every day using the appropriate MEF conditioned SILAC light or heavy ES Cell medium Passage the cells at a ratio of 1 3 to 1 6 when cells reach 70 80 confluency 4 7 days Expand each cell population for at least six doubling times usually is about 2 weeks to achieve gt 90 incorporation of labeled amino acid into the proteins After six doublings harvest a small aliquo
74. ring only Not intended for human or animal therapeutic or diagnostic uses Coomassie is a registered trademark of Imperial Chemical Industries PLC Explorer ProteinPilot Voyager DE and MALDI TOF TOF are trademarks and QSTAR OTRAP are registered trademarks of Applied Biosystems MDS Sciex a joint venture between Applera Corporation and MDS Inc ICAT is a registered trademark of the University of Washington and is exclusively licensed to the Applied Biosystems Group of Applera Corporation LCQ LTQ LTQ FT and BioWorks are trademarks of Thermo Fisher Scientific Inc Matrigel is a trademark of Becton Dickinson and Company Q Tof and ProteinLynx Global SERVER are trademarks of Waters Corporation SpeedVac is a registered trademark of Thermo Savant Inc 54
75. room temperature 12 Vortex for 30 seconds centrifuge at 14 000 x g for 1 minute and collect the supernatant 13 Add 50 ul 5 FA 50 acetonitrile and incubate for 30 minutes at room temperature 14 Vortex for 30 seconds centrifuge at 14 000 x g for 1 minute and collect the supernatant pooling it with the supernatant from Step 12 15 Concentrate the supernatant using a centrifugal vacuum concentrator to 5 ul Do not allow the samples to dry out 16 Submit your tryptic peptides to the proteomics core facility for analysis 49 Technical Support World Wide Web Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3
76. s for MS analysis protein identification and quantitation e Troubleshooting Description of Kit Components Kit Components SILAC KnockOut D MEM KnockOut Serum Replacement GlutaMAX I Supplement The SILAC Stem Cells Protein ID and Quantitation Media Kit includes the following major components e GIBCO KnockOut D MEM for growth of human or mouse embryonic stem cells e GIBCO KnockOut Serum Replacement for efficient and reproducible ES cell growth without any interfering amino acids for SILAC e SILAC Normal light amino acids for supplementing the basal medium for cell culture e SILAC Isotope labeled heavy amino acids for performing isotope labeling in cell culture The SILAC KnockOut D MEM is a basal medium optimized for growth of undifferentiated ES cells The osmolarity is optimized to approximate that of the murine embryonic environment The SILAC KnockOut D MEM is a basal medium that requires supplementation with amino acids GlutaMAX I and KnockOut Serum Replacement for cell culture For feeder free culture the medium can be conditioned in the presence of MEF cells see page 13 for preparing media The SILAC KnockOut D MEM is formulated without L Arginine L Glutamine L Lysine and phenol red TM The Knockout Serum Replacement is a serum free formulation optimized to grow and maintain undifferentiated ES cells in culture Goldsborough et al 1998 It dir
77. se No 5 Invitrogen Technology Limited Use Label License No 161 SILAC Limited Use Label License No 297 Media for Stem Cell Culture 52 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not
78. se the instrument software to perform protein identification TM The protein identification method for SILAC kits was developed by processing the raw MS data files from MS with Mascot Distiller Matrix Science London and then searched the NCBI database using Mascot search algorithm Our results have shown that using Mascot to identify proteins provides 40 better results than compared to other protein identification methods Certain MS instruments contain software that perform protein identification using the Mascot search algorithm For example the GPS Explorer 3 0 software with AB MDS Sciex Family of MALDI TOF TOF Analyzers For more information on Mascot Distiller visit www matrixscience com Continued on next page Protein Identification and Quantitation continued Using Mascot for Protein Identification Brief instructions are provided below to set up the Mascot server settings for protein identification using GPS Explorer Similar procedures apply to stand alone versions of Mascot server For installation set up and detailed instructions on using Mascot visit www matrixscience com 1 Start GPS Explorer software on your MS instrument AB MDS Sciex Family of MALDI TOF TOF Analyzers Start Mascot server on your local computer and navigate to the Mascot Modification File screen Mascot Configuration Mascot Modification Files gt A Error tolerant search substitutions Microsoft Hardware 4
79. t variance amongst the peptides correlated to the same protein may indicate the following e The protein identification was incorrect e Co elution of an unrelated isobaric peptide distorted the peak profile e Certain residues occurring in the peptide outlier are subject to metabolic interconversion for example Arg to Pro Under these conditions check the profile manually and dismiss the peptide from the analysis if appropriate 35 Troubleshooting TM Introduction Review the table below to troubleshoot your experiments using SILAC Stem Cells Protein Identification and Quantitation Kits For troubleshooting MS refer to the manual supplied with the MS instrument or contact the core facility Protein ID scores are Insufficient cells used Use the recommended number of cells for each low or poor data quality application as described on page 15 Use 5 fold after MS more cells to obtain a good signal after MS for low abundant proteins Improper MS analysis Ensure the MS instrument was properly tuned and calibrated prior to sample analysis Check that the correct database organism taxonomy peptide modifications labeled amino acid and enzyme were selected during data analysis Loss of phosphopeptides Follow the manufacturer s instructions to properly after enrichment step prepare the phosphopeptide column and perform chromatography to avoid any loss of phosphopeptides Loss of peptides after Do not concentrate the
80. t of cells 1 x 10 cells from each cell population to determine the efficiency of incorporation Store the cell pellet at 80 C At the end of six doublings you will have 6 4 x 10 cells when starting with 1 x 10 cells for each cell population See page 15 for the approximate number of cells needed for each analysis Perform the appropriate cell treatment page 21 and process samples page 23 Analyze tryptic peptides using MALDI TOF MS or LC MS Perform protein identification using MS instrument software or Mascot software suite Quantitate protein using MS instrument software Kit Contents and Storage TM Shipping and The shipping and storage conditions for SILAC Stem Cells Protein Storage Identification ID and Quantitation Media Kit with U C L Lysine Lys and KnockOut D MEM are listed below For a detailed description of kit components see page 4 The kit includes appropriate media components and amino acids Store all media protected from light Box Component Shipping Storage 1 SILAC KnockOut D MEM Room 2 8 C temperature KnockOut Serum Replacement Dry ice 5 to 20 C GlutaMAX I Supplement 100X Dry ice 5 to 20 C MEM Non Essential Amino Acids Solution Gel ice 2 8 C 10 mM 100X liquid SILAC Phenol Red Solution 10 g L Gel ice 2 8 C SILAC L Lysine HCl and L Arginine Gel ice 2 8 C SILAC U C L Lysine HCl Lys Gel ice 2 8 C 2 mercapt
81. tails on interpreting the results fei GPS Explorer TM Software Results Browser File Edit View Help t 9 t id A o Next Up Projects Properties Refresh Spotfire GPS Explorer TM Software admin Dd amp Hk RB BI Protein Spectrum Prev Spec Next5pec DBResul Help o 9 Accession Total lon Best lon Humber Siem MW Protein Pt Score Cl Score C l oxygen regulated protein precurs gil5453832 111266 2 5 1600 100 000 100 000 coatomer protein complex subur gi4758030 1382433 7 7000 100 000 38317 unnamed protein product Homo gi28678 10934747 52400 3 100 000 100 000 Tripeptidyl peptidase Il TPPI gi34223721 1382626 5 9000 100 000 88 250 bAG2C3 2 isoleucineRNA syntl gil2314134 1455066 58600 5 100 000 33 543 eukaryolic translation initiation fa gi4503508 166468 3 amp 3800 10 100 000 99 990 VLA 3 alpha subunit Homo sapii gi220141 1134334 B1300 4 100 000 98 606 integrin beta 1 isoform 1A precur giS743813 883570 52700 5 100 000 94 580 bA462D18 32 ribosome bindinc gii4149066 152097 5 87300 3 100 000 99 998 unnamed protein product Homo gil16552261 47458 9 5 0100 5 100 000 94 298 3 3 4 3 Protein Name ajajajaja ABP125 Homo sapiens gigoos4s0 1289720 amp 8000 100 000 99 998 100 000 33 587 100 000 99 855 100 000 99 958 Scaffold attachment
82. this application you need 60 x 10 cells or 2 x 100 mm plate of cells from each cell population 1 10 11 Harvest light and heavy labeled culture media separately after stimulation Step 4 page 21 by centrifugation The volume of the medium is usually 20 ml if using 2 x 100 mm culture plates Filter each media using a 0 45 um filtration device to remove any cell debris Concentrate each media to about 0 5 ml using an ultrafiltration device with a 5000 Da molecular weight cut off membrane Determine the protein concentration in each sample Mix equal amount of proteins from media sample labeled with light and heavy amino acids Dry the media sample in a centrifugal vacuum concentrator e g Thermo Savant SpeedVac centrifuge Resuspend the media sample in 60 200 ul 1X NuPAGE LDS Sample Buffer containing 1X NuPAGE Reducing Agent Heat the samples at 70 C for 8 10 minutes Separate the proteins by SDS PAGE using NuPAGE Novex precast gels page 47 Transfer stained gel to the proteomics core facility or proceed to the next step Perform in gel trypsin digestion page 49 Proceed directly to MS analysis after trypsin digestion page 27 Submit your tryptic peptides to the proteomics core facility for analysis After data analysis perform crossover experiments to verify the growth factors identified see Important below We recommend that you verify the growth factors identified in the initial experiments by
83. tion Proc Nat Acad Sci 96 6591 6596 Continued on next page 53 References Continued Ong S E Blagoev B Kratchmarova I Kristensen D B Steen H Pandey A and Mann M 2002 Stable Isotope Labeling by Amino Acids in Cell Culture SILAC as a Simple and Accurate Approach to Expression Proteomics Molecular and Cellular Proteomics 1 376 386 Peter J 2000 Proteome Research Mass Spectrometry Peters J Ed Springer Verlag Berlin Sechi S and Oda Y 2003 Quantitative Proteomics Using Mass Spectrometry Curr Opin Chem Biol 7 70 77 Shevchenko A Wilm M Vorm O and Mann M 1996 Mass Spectrometric Sequencing of Proteins from Silver stained Polyacrylamide Gels Anal Chem 68 850 858 Simpson R J 2003 Proteins and Proteomics A Laboratory Manual Cold Spring Harbor Press New York Speicher D 2004 Proteome Analysis Interpreting The Genome Speicher D Ed Elsevier Press Oxford Xu C Inokuma M S Denham J Golds K Kundu P Gold J D and Carpenter M K 2001 Feeder free Growth of Undifferentiated Human Embryonic Stem Cells Nature Biotech 19 971 974 Zhu H Hunter T C Pan S Yau P M Bradbury M E and Chen X 2002 Residue specific Mass Signatures for the Efficient Detection of Protein Modifications by Mass Spectrometry Anal Chem 74 1687 1694 2005 2006 Invitrogen Corporation All rights reserved For research use further cell culture manufactu
84. to process the samples after labeling If you have an established protocol to process samples for MS analysis for a specific application use the established protocol e Recipes for lysis buffer and protocols for cell lysate to analyze phosphoproteins or study membrane proteins are described on page 43 e General methods for protein analysis such as immunoprecipitation analyzing protein complexes and SDS PAGE are described on page 47 General guidelines and workflows for the following applications are described in this manual e Identifying growth or differentiation factors e Analyzing differential protein expression or identifying unique cell surface markers upon differentiation e Studying signal transduction pathway i e changes in phosphorylation lipidation relocation ubiquitination upon stimulation Depending on the analysis that you wish to perform with the ES cell samples choose the appropriate sample processing workflow and guidelines to obtain the best results Note The SILAC Stem Cells Kit is not limited only to the above listed applications After labeling the ES cell samples can also be used for other applications such as protein and or peptide arrays Continued on next page 23 Processing Samples Continued Identifying Growth The general workflow for identifying growth or differentiation factors using ES cell labeling medium is described below or Differentiation Factors Important 24 For
85. topic Labeling by Amino Acids in Cell Culture Stem Cells Protein Identification ID and Quantitation Media Kit provides a simple efficient and reproducible method for quantitative analysis of differential protein expression in human or mouse embryonic stem ES cells The kit is designed to allow efficient metabolic labeling of embryonic stem cells followed by sample preparation and analysis using mass spectrometry MS The SILAC technology is a powerful tool for quantitative analysis of systems biology including post translational modifications low abundance proteins phosphoproteins and membrane proteins using mammalian cells The SILAC Protein ID and Quantitation Kits are based on the metabolic labeling technology developed by Brian Chait Oda et al 1999 using isotopic nutrients N in cell culture media and performing comparative MS analysis Chen and coworkers modified this method by using stable isotopes of amino acids instead of simple salts Chen et al 2000 Because isotopic amino acids are incorporated into proteins in a sequence specific manner with Amino Acid Coded mass Tags AACT they were able to confirm the identity of proteins with higher confidence by comparing the sequence dependent mass shifts of an entire protein digest to the peptide mass fingerprint of the unlabeled protein Residue specific mass alterations to efficiently detect protein modifications such as phosphorylation and oxidation were also demonstrated using
86. twice with a suitable buffer to remove any nonspecific proteins 11 Resuspend the pellet in 50 ul 1X NuPAGE LDS Sample Buffer Add 5 ul NuPAGE Sample Reducing Agent 10X 12 Heat the sample at 70 C for 8 10 minutes 13 Centrifuge the sample for 1 minute at 10 000 x g and load supernatant onto a NuPAGE Novex Bis Tris Gel and analyze the protein immune complexes using SDS PAGE next page Continued on next page 46 General Methods for Protein Analysis Continued Analyzing Protein Instructions for analyzing protein complexes in solution using protein specific Complexes antibodies and Protein G Agarose are described below You may use Protein A beads if desired 1 Tothelysate prepared as described on pages 24 26 add 30 50 ug of the bait protein that allows binding to the protein complex Note You may optimize the amount of protein used based on the initial results 2 Add 20 50 ul epitope tagged resin such as GST agarose or Streptavidin agarose to precipitate the protein complex if your protein of interest contains an expressed GST tag or a biotin tag 3 Rock for 2 24 hours at 4 C 4 Centrifuge at 10 000 x g for 5 minutes at 4 C Remove supernatant 5 Washthe pellet twice with a suitable buffer to remove nonspecific proteins 6 Resuspend the pellet in 16 20 ul 1X NuPAGE LDS Sample Buffer and add 2 ul of NuPAGE Sample Reducing Agent 10X 7 Heatthe sample at 70 C for 8 10 minutes Centrifuge the sample for 1 mi
87. x by immunoprecipitation page 46 Separate the immunoprecipitated proteins by SDS PAGE using NuPAGE Novex precast gels page 47 Transfer stained gel to the proteomics core facility or proceed to the next step Perform in gel trypsin digestion page 49 Proceed directly to MS analysis after trypsin digestion page 27 Submit your tryptic peptides to the proteomics core facility for analysis Mass Spectrometric Analysis Introduction Important General Guidelines Recommended Methods for MS Analysis General guidelines for performing MALDI TOF MS and LC MS analysis of tryptic digested peptides page 49 are described in this section For details on the use of various MS instruments for analysis refer to the manual supplied with the instruments This section is designed for experienced users of MALDI TOF and LC MS analysis especially core facility personnel that are familiar with standard techniques and instruments for MS analysis General recommendations are included but detailed protocols for using the MS instruments are not included Basic guidelines for sample preparation are given below The choice of matrix and the amount of sample needed for mass spectrometry analysis depends on the technique used for analysis and the individual protein sample For more details on sample preparation contact your proteomics core facility For more information refer to published protocols Ausubel et al 1994 Coligan et al 199
88. y differential expression see Important previous page If you have performed any type of cell treatment be sure to lyse the control cells from Step 2 page 21 using the same lysis method used for treated cells Continued on next page 25 Processing Samples Continued Studying Signal Transduction Pathway 26 The general workflow for studying signal transduction pathway using labeled ES cells is described below For this application you need 107 108 cells or 2 to 3 x 100 mm plate of cells from each cell population After labeling for six doubling times and performing the cell treatment if appropriate harvest cells from each ES cell population as below 1 10 11 Determine the viable and total cell count on an aliquot of cells using the trypan blue method Harvest the required number of ES cells from each population page 21 Resuspend each cell pellet in 1 ml chilled PBS Mix the cells grown in light normal medium and heavy isotope labeled medium in a 1 1 ratio based on the cell number Centrifuge the cells at 500 x g for 5 minutes at 4 C to remove PBS Resuspend the cell pellet and lyse the cells in a non ionic detergent containing buffer Be sure to use appropriate protease inhibitors in the buffer See page 43 for lysis buffer recipes and protocols to prepare cell lysate Mix well by pipetting up and down Use the lysate to perform affinity enrichment of the signaling molecule or protein comple
89. y of water and seek medical advice Wear suitable protective clothing See MSDS for details e The 2 Mercaptoethanol 1000X is toxic Do not breathe vapor and avoid contact with skin and eyes Do not mouth pipet the solution See MSDS for details Continued on next page 11 Preparing Cells and Media Continued Materials Needed Components Supplied in the Kit Media Types 12 You need the following items e Human or mouse ES cell lines available from commercial sources or ATCC e Human bFGF page vii for human ES cells e Murine Leukemia inhibitory factor Millipore catalog no ESG1106 for mouse ES cells e Regular ES Cell Medium see below e Optional growth factors if needed for your cells e Appropriate tissue culture dishes and media bottles e 37 C incubator with a humidified atmosphere of 5 CO in air e Sterile centrifuge tubes e Reagents to determine viable and total cell counts page vii e 0 22 um filtration unit to filter sterilize the medium e Optional U 9C Na L Arginine or U C L Arginine for double labeling experiments page vii You need the following items supplied with the kit e SILAC KnockOut D MEM deficient in lysine arginine glutamine and phenol red e KnockOut Serum Replacement thaw and store at 4 C until use or aliquot and freeze at 5 to 20 C thaw as needed e L Lysine HCl e L Arginine e GlutaMAX I Supplement 100X thaw and store at 4 C until use e SI

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