Name: Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae (Video) | JoVE Journal
Uploaded: 2009-10-12T23:00:00.000+00:00
Duration: 15 min 41 s
Description: Learn quantitative phosphoproteomics in Saccharomyces cerevisiae using cryolysis, HILIC fractionation, and IMAC enrichment. This method enables precise analysis of cellular signaling mechanisms via mass spectrometry.

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5 minutes

We would like to thank Dr. Rich Rogers for technical assistance with mass spectrometry analyses and helpful discussions, and Drs. Rob Moritz, Jeff Ranish, and Hamid Mirzai for helpful discussions.
This work was funded by the NIH Centers of Excellence.

Cell growth and media
<ol>
 <li>A single colony of BY4742&Delta;arg4&Delta;lys1 cells overnight in 100 mL rich media to an OD600 of 1.0 then seed into two 1 liter cultures of a minimal yeast medium (0.17% Yeast Nitrogen Base without Ammonium Sulfate or Amino Acids, 0.5% Ammonium sulfate) containing a full complement of amino acids, supplemented with 20mg/L of isotopically normal or heavy arginine (13C6 15N4; Isotec) and lysine (13C6 15N2; Isotec).</li>
 <li>The cells were grown for 18 hours, to an OD600 of 1.8. It is critical that the cells go through at least 9 generations to achieve full incorporation of the labeled isotopes. The light sample was pelleted, washed with sterile water, reseeded into an oleate containing medium (isotopically normal arginine and lysine, 0.2% Oleate (Sigma Chemicals) and 0.5% Tween 40 (Sigma Chemicals)) and stimulated for another 85 minutes. This yields an isotopically light, with respect to arginine and lysine, oleate-stimulated sample and an isotopically heavy glucose-grown reference sample.</li>
</ol>
Cell Lysis, Isolation and Fractionation of Peptides
<ol start="3">
 <li>Weigh centrifuge bottle. Harvest samples by centrifugation for 3 minutes. Remove media and aspirate excess liquid. Weigh pellet and bottle (will typically yield between 1-2 grams) then flash freeze in liquid nitrogen. Add a volume equivalent to pellet weight of 'grinding' buffer to the frozen pellets in liquid nitrogen (Phosphate Buffered Saline (PBS, Gibco), 10% glycerol, Protease Inhibitors (SigmaFAST Protease Inhibitor Tablets, Sigma) and HALT Phosphatase inhibitors (Thermo Scientific)).</li>
 <li>Freeze the grinding vessel in liquid nitrogen. Wait until the liquid nitrogen ceases boiling. Transfer the pellet to the grinding vessel, with frozen ball bearings.</li>
 <li>Grind at 600 rpm with a 1 min 20 sec cycle with a direction reversal for 3 minutes. Refreeze the grinding vessel in liquid nitrogen. Repeat 4 more times for 15 minutes total grinding time.</li>
 <li>Collect the frozen grindate into a 50ml Falcon tube place dry ice. Store at -80 &deg;C until ready to use.</li>
 <li>Make a 10ml solution of 8M urea, 0.1M ammonium bicarbonate, 0.1M Tris pH 8.6. Add 3 volumes of the urea buffer to 1 volume of the frozen grindate (for example 3mls of the urea buffer added to a 1 gram pellet with 1 ml of PBS buffer). Immediately sonicate the mixture with a probe tip sonicator (2 - 10 second pulses). Keep the tip near the bottom of the tube to prevent foaming of the solution. The grindate should immediately go into solution.</li>
 <li>Clear the lysate by centrifugation for 5 minutes at 4&deg;C.</li>
 <li>Transfer supernatant to fresh tubes.</li>
 <li>Make a fresh aliquot of 0.5M Tris[2-carboxyethyl] phosphine (TCEP). Add to sample at 1:100 for a 5mM final concentration. Incubate at 37&deg;C for 1 h.</li>
 <li>Alkylation of reduced cysteines. Allow to cool to room temperature. Make a fresh aliquot of 1M iodoacetamide. Keep in the dark (we wrap the tube in foil). Add to a final concentration of 20mM. Incubate in the dark for 1 h at room temperature.</li>
 <li>Quench the iodoacetamide with 20mM DTT from a 1M stock at room temperature for 1 h.</li>
 <li>Quantify the protein with a standard Bradford or BCA assay (not described). Take a sample for analysis by SDS PAGE below.</li>
 <li>To validate incorporation of the heavy isotopes, use 100 &mu;l of the reduced and alkylated lysate. Dilute 1:4 with dH2O, sonicate and add trypsin at 50:1 protein to trypsin. Digest for a minimum of 4 hours, dry sample down in a speed vac and then desalt with a C18 Ultramicrospin columns (The Nest Group).
 <ol>
 <li>Hydrate column with 100 &mu;l of Acetonitrile</li>
 <li>Equilibrate with 200 &mu;l 0.1% TFA.</li>
 <li>Resuspend dry sample in 200 &mu;l 0.1% TFA. Load onto column. Centrifuge at ~200 x g or the minimal speed required for flow through the column.</li>
 <li>Wash twice with 200 &mu;l 0.1% TFA.</li>
 <li>Elute with twice with 50 &mu;l 60% Acetonitrile, 0.1% TFA.</li>
 <li>Dry sample in a speed vac.</li>
 <li>Resuspend sample in 20 &mu;l 0.1% Formic acid. Run 2 &mu;l on a mass spectrometer. Check incorporation of isotopically heavy arginine and lysine by a 10 and 8 Dalton shift in the respective m/z spectra. The incorporation for each amino acid should be between 96 - 98%.</li>
 </ol>
 </li>
 <li>Mix equivalent amounts (mg of proteins) of the isotopically heavy and light samples.</li>
 <li>Dilute sample 1:4 with 20% methanol.</li>
 <li>Add trypsin at 1:200. Incubate overnight at 37&deg;C.</li>
 <li>Check that digestion has gone to completion by SDS PAGE. Run 2 and 4 &mu;l of the predigestion sample and 8 and 16 &mu;l of the post trypsin digest. Visualize by Coomassie staining that digestion is complete. If it is not complete, sonicate the sample with a probe tip sonicator briefly and digest again with trypsin.</li>
 <li>Dry the sample down in a speed vac. When dry add 200 &mu;l of filtered dH2O and vortex till the pellet goes into solution. Dry sample down in a speed vac. Resuspend again with dH2O. Dry sample again.</li>
 <li>Resuspend pellet in 0.1% Trifluoracetic Acid (TFA; Sigma). Check that the pH is around 3 using pH strips. If needed acidify with 10% TFA.</li>
 <li>Pellet out any precipitate.</li>
 <li>Desalt the samples on a Waters Sep-Pak Vac 500 mg C18 column. Do not overload these columns. The maximum amount is 5 mg but it's better to load a little less. Divide the sample between multiple columns if necessary.
 <ol>
 <li>Hydrate the column with 5 ml 100% acetonitrile. Centrifuge at ~200 x g or the minimal speed required for flow through the column.</li>
 <li>Equilibrate column with 5ml 0.1% TFA. Load sample. Collect flow and load again.</li>
 <li>Wash with 5 ml 0.1% TFA. Repeat.</li>
 <li>Elute with 2ml 60% Acetonitrile, 0.1% TFA.</li>
 <li>Dry sample down in a speed vac.</li>
 </ol>
 </li>
 </ol>
 Peptide Fractionation and Isolation of Phosphopeptides
<ol>
<li>Peptides are fractionated on a HILIC column. We use a TOSOH TSK-Gel Amide-80 4.6 mm x 25 cm analytical column with a guard column. Take your time with this step, run the column for 2 hours at 90% A then run two blank gradients before loading your sample. The maximum load on a column this size is thought to be around XX, we typically do not load more than 5 mg per run.</li>
<li>Solvent A - 98% ACN/0.1% TFA 
Solvent B - 2% ACN/0.1% TFA 
Load in 90% A over 20', 90% A to 85% A over 5', 85% A to 60% A over 40', 60% A to 0% A over 5', 0% A to 90% A over 5'.</li>
<li>The flow rate is 1ml/min and fractions were collected at 2 minutes intervals beginning at the 85% A to 60% A gradient.</li>
<li>Combine fractions to reduce the number of fractions to 10. Increased fractions will likely yield greater coverage of the phosphoproteome.</li>
</ol>
Enrichment of phosphopeptides
<ol>
<li>Phosphopeptides are enriched using PHOS-Select Iron Affinity Gel. We aliquot our IMAC resin to avoid repeated freeze thawing. Make 50 mls of a 250 mM acetic acid, 30% ACN load solution. Bring the pH to 2.7 with HCl. Resuspend pellets in 500 &mu;l of load solution. Check pH, adjust with HCl or NaOH as necessary.</li>
<li>Prewash 200 &mu;l of IMAC beads in an appropriate column. We use Molbiotec spin columns. 
Add 15 &mu;l of a 50% slurry (gel to load solution) to each fraction, and incubate the samples for 30' with end over end rotation. Keep the flow for analysis.</li>
<li>Wash samples 3 times with load solution and once with 500 &mu;l filtered dH2O.</li>
<li>Elute peptides with 2- 3 min with 400 &mu;l of 50 mM Na2HPO4 (pH 8.4) buffer. Transfer to a glass vial.</li>
<li>Acidify to pH 3 with 5 &mu;l 100% formic acid, flash freeze in liquid nitrogen, and dried sample down in a speed vac. </li>
<li>Resuspend phosphopeptides in 200 &mu;l of 0.1% TFA and C18 clean as described above. Dry down eluted samples in a speed vac. </li>
<li>Resuspend peptides in 20 &mu;l of 0.1% formic acid. Use 2 &mu;l per mass spectrometer analysis or as needed.</li>
</ol>
Notes
For much of this protocol you will be working 'blind'. The cell lysis can be monitored by western blotting and Bradford or BCA assays, and the trypsin digestion can also be readily assessed. The HILIC column will gave a trace similar to this one, or as seen by others [1, 2] depending on the liquid chromatography machine that is used. Samples are monitored by mass spectrometry; test your samples on inexpensive machines such as LCQs or MALDIs before going to the high cost analyses on a high mass accuracy, high resolution machine. Finally if you a drying down a sample with acid in it, use glass.

<ol><li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li>
<li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li>
<li>Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li>
<li>Ross, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li>
<li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li>
<li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphoproteome+analysis+of+fission+yeast%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li>
<li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+SCX%2FIMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li>
<li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques%5BTitle%5D)+AND+%22Rapid+Commun+Mass+Spectrom%22%5BJournal%5D)">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li>
<li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li>
<li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS%2FMS+and+titanium+oxide+precolumns%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li>
<li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li>
<li>Zhou, H., Watts, J. D., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+approach+to+the+analysis+of+protein+phosphorylation%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">A systematic approach to the analysis of protein phosphorylation.</a> Nat Biotechnol. 19 (4), 375-378 (2001).</li>
<li>Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducible+isolation+of+distinct%2C+overlapping+segments+of+the+phosphoproteome%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Reproducible isolation of distinct, overlapping segments of the phosphoproteome.</a> Nat Methods. 4 (3), 231-237 (2007).</li>
</ol>

This method will yield an enrichment of phosphopeptides that can be quantitatively analyzed. A number of parameters can be altered in this protocol, but the most important aspect to remember is to preserve your phosphorylation. Preparation of cells for quantification can be achieved in a number of different ways, for example if you cannot label your cells in vivo, tags such as ICAT[3] or ITRAC [4] can be used post extraction to differentially label two cultures for quantification purposes. 
<p class="jove_c...

<table><tbody><tr><td>Isotec&amp;#8482; L-Arginine-&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;,&lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>608033</td><td></td></tr><tr><td>Isotec&amp;#8482; L-Lysine-&lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>609021</td><td></td></tr><tr><td>GIBCO&amp;#8482; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td><td>Invitrogen</td><td>70011044</td><td></td></tr><tr><td>SigmaFAST Protease Inhibitor Tablets</td><td>Sigma-Aldrich</td><td>S8820</td><td></td></tr><tr><td>Pierce Halt Phosphatase Inhibitor Cocktail</td><td>Thermo Fisher Scientific, Inc.</td><td>78420</td><td></td></tr><tr><td>Retsch PM100 Ball Mill Grinder </td><td>Retsch</td><td>20.540.0001</td><td></td></tr><tr><td>Retsch 125 ml Stainless Steel Grinding Jar</td><td>Retsch</td><td>01.462.0148</td><td></td></tr><tr><td>Ultramicrospin C18 column</td><td>The Nest Group</td><td>SUM SS10</td><td></td></tr><tr><td>Sep-Pak Vac 500 mg C18</td><td>Waters</td><td>WAT043395</td><td></td></tr><tr><td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td><td>Tosoh Corp.</td><td>13071</td><td>This is the HILIC chromatographic column.</td></tr><tr><td>Guard column 4.6 mm ID x1 cm</td><td>Tosoh Corp.</td><td>19021</td><td>We recommend this guard column to extend the life of your HILIC column.</td></tr><tr><td>PHOS-Select&amp;#8482; Iron Affinity Gel</td><td>Sigma-Aldrich</td><td>P9740</td><td>This is the IMAC resin. Aliquot and store.</td></tr></tbody></table>

quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Phosphorylation is a key cellular signaling mechanism governing processes from protein localization to DNA transcription and the study of the phospho alone by mass spectrometry is an exciting new advance in the study of both signaling and proteomics. Differentially labeled cells are lies by lysis in a ball mill grinder, then reduced alkylated mixed and digested. The digested peptides are fractionated by hydrophilic interaction chromatography and phospho peptides enriched from fractions by iMac enrichment.The phospho peptide enriched fractions are then identified by msm. Ms.Hi, I'm Ramsey Celine from the E group at the Institute Persistence Biology. Today we're gonna show you a procedure for the extraction of metabolically labeled phospho peptides.We use this procedure in our laboratory to quantitatively study the fossa proteome. So let's get started. This procedure begins with growing isotopically light and heavy yeast cells.First inoculate 100 milliliters of rich media with a single colony of yeast cells. Grow this culture overnight to an OD 600 of 1.0. Then use it to seed two one liter cultures of minimal medium containing a full complement of amino acids and supplemented with 20 milligrams per liter of isotopically.Normal or isotopically heavy arginine and lysine grow both one liter cultures for 18 hours to an OD 600 of 1.8. It is critical that the cells go through at least nine generations to achieve full incorporation of the labeled isotopes. When the cells have fully incorporated the labeled isotopes centrifuge, the cells that were grown in the media with isotopically, normal arginine and lysine.To obtain a cell pellet, wash the pellet with sterile water sede into an oleate containing medium with isotopically, normal arginine and lysine and stimulate for 85 minutes. This yields an isotopically light oleate stimulated sample. The other culture grown in media with isotopically heavy arginine and lysine is not stimulated with oleate and will serve as an isotopically heavy glucose grown reference sample.To collect one liter, the culture is distributed between four 250 milliliter centrifuge bottles. After centrifugation cells are collected into one tube by removing or but 100 milliliters of the media Resus suspending the cells and transferring to the next tube. At the end, one large pellet is collected in a single tube centrifuge for three minutes at 4, 000 RPM.After removing media, the centrifuge bottle and pellet weight is compared to an empty centrifuge bottle. After centrifugation, remove media and aspirate excess liquid from the bottles, weigh the pellets and a typical yield is between one to two grams. Next, flash freeze The pellets in liquid nitrogen add grinding buffer that contains protease and phosphatase inhibitors to the pellets.The volume of grinding buffer added should be equivalent to the pellet weight. Freeze the grinding vessel in liquid nitrogen. When the liquid nitrogen has ceased boiling.Use a scapula to chip away at the pellet and transfer it to the grinding vessel that contains frozen ball bearings. Grind the pellets at 600 RPM with a one minute 22nd cycle with a direction reversal for three minutes. Refreeze the grinding vessel in liquid nitrogen.Repeat the grinding four more times for a total of 15 minutes. Grinding time when the grinding is complete for both samples. Collect the frozen grind date into 50 milliliter falcon tubes and keep frozen either in liquid nitrogen or in dry ice.Store the grind date at minus 80 degrees Celsius until ready for use. Isolation and fractionation of the peptides begin with their alkylation. The first step is to solubilize your sample in the urea solution.Bring each grind date into solution by adding three volumes of urea buffer to one volume of frozen grind date. For example, three milliliters of RIA buffer is added to a one gram pellet. With one milliliter of PBS buffer.Immediately sonicate the mixture with the probe. Tip sonicate. Keep the tip near the bottom of the tube to prevent foaming of the solution.The grind date should immediately go into solution. Clear the lysates by centrifugation at 6, 000 RPM for five minutes. At four degrees Celsius, transfer the SUPINATE to fresh tubes.To begin the reduction reaction, add freshly prepared 0.5 molar tris, two carboxyl LITAL PHOSPHINE or TCEP to each sample at a one to 100 dilution. To achieve a final TCEP concentration of five millimolar, incubate the samples at 37 degrees Celsius for one hour. After the 37 degrees Celsius incubation, allow the samples to cool to room temperature.Then add freshly prepared one molar io acetamide to a final concentration of 20 millimolar incubate in the dark for one hour at room temperature. Following the one hour incubation, quench the iota acetamide with 20 millimolar DTT. Leave it room temperature for one hour.The reduced and alkylated lysates are now ready for tripsin digestion. At this point, samples of the lysates can be used for protein quantification SDS page analysis to verify cell lysis and mass spectrometry analysis to validate incorporation of the heavy isotopes. To begin tripsin digestion, mix equivalent amounts of the isotopically heavy and light samples.Dilute the sample one in four with 20%methanol and add trypsin at a one in 200 dilution. Incubate the sample overnight at 37 degrees Celsius on the following day. After verifying complete digestion by SDS page and kumasi staining, dry the sample down in a speed vac when the sample is dry.At 200 microliters of filtered distilled water and vortex until the pellet goes into solution. Dry the sample down in a speed vac and resuspend it again with distilled water After drying the sample, once again, resus resuspend the pellet in 0.1%Tri Fluor acetic acid or TFA, check that the pH is around three using pH strips if needed. Acidify with 10%TFA pellet out any precipitate.The tryin digested sample will now be desalted using a water set pack VAC 500 milligram C 18 column. To begin, hydrate the column with five milliliters, 100%aceto nitrile centrifuge at around 200 times G or the minimal speed required for flow through the column. Next, equilibriate the column with five milliliters, 0.1%TFA load the sample.Be careful not to overload the column and if necessary, divide the sample between multiple columns. Collect the flow into a waste container and load the column again if necessary, wash with five milliliters, 0.1%TFA. Repeat this wash elute with two milliliters of 60%Aceto nitrile 0.1%TFA EEU must be collected by centrifugation.Dry the sample down in a speed vac resuspend the dried sample in 200 microliters of solvent B.The resuspended sample is now ready for peptide fractionation. Fractionate the peptides on A TSK gel AMIDE 80 analytical column with a guard column. Take your time with a step.Run the column for two hours at 90%solvent A then run two blank gradients before loading your sample. We typically do not load more than five milligrams per run on a column of this size, modern and even not so modern liquid chromatography. Machines will allow for automated sample injection fraction collection flow, rate control, and gradient mixes.These are set at the beginning of the run as described. Load in 90%A over 20 minutes, 90%A to 85%A over five minutes, 85%A to 60%A over 40 minutes, 60%A to 0%a over five minutes and 0%A to 90%a over five minutes. Set the flow rate at one milliliter per minute and collect fractions at two minute intervals.Beginning at the 85%A to 60%A gradient combine fractions to reduce the number of fractions to 10 increased fractions will likely yield greater coverage of the phospho proteome. Phospho peptides are enriched by immobilized metal chromatography. We use the phos select iron affinity gel as the iMac resin.First Resus suspend pellets in 500 microliters of load solution. Next, add 15 microliters of a 50%slurry to each fraction and incubate the samples for 30 minutes with end over end rotation. Keep the flow for analysis.Wash the sample three times with load solution and once with 500 microliters filtered distilled water after the washes, elute peptides for two to three minutes with 400 microliters of 50 millimolar of sodium phosphate buffer. Transfer the eluted peptides to a glass vial. Add five microliters, 100%formic acid to the peptides to acidified to pH three resuspend phospho peptides in 200 microliters of 0.1%TFA and desalt again with a C 18 column as described previously, dry down the alluded samples in a speed vac.Finally resuspend the phospho peptides in 20 microliters of 0.1%formic acid. The samples are now ready to be quantitatively analyzed by mass spectrometry. A chromatic graphic trace from the fractionation of total cellular peptides using H-I-L-I-C chromatography is shown here.It can be observed in this trace that while phospho peptides are found throughout the elucian profile, the bulk of the phosphorylated residues are retained until near the end of the elucian profile. We've just shown you how to grind cells, digest proteins, fraction a peptides, and enrich for phospho peptides using immobilized metal affinity chromatography. When doing this procedure, it's important to remember to preserve phosphorylation and check the pH's Beer solutions.So that's it. Thanks for watching and good luck with your experiments.

quantitative-phosphoproteomics-fatty-acid-stimulated-saccharomyces

Research

JoVE Journal

Biology

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identification of protein complexes with quantitative proteomics in S. cerevisiae

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS).  This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Here, we introduce a protocol for using yeast surface-displayed substrates for enzymatic modification assays. The platform was demonstrated using the analysis of the dephosphorylation activity of tyrosine phosphatase SHP-2 against one of its substrates as a representative enzymatic modification assay.

Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

Learn quantitative phosphoproteomics in Saccharomyces cerevisiae using cryolysis, HILIC fractionation, and IMAC enrichment. This method enables precise analysis of cellular signaling mechanisms via mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

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