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.
