The overall goal of the following experiment is to profile methyl transferases from complex protein mixtures by capture compound mass spectrometry or CCMS. The methyl transferases are functionally isolated by tri functional capture compounds that carry the general methyltransferase product inhibitor, S adenyl, L homocystine short, SAH as the selectivity function in the workflow shown here, the functional isolation is achieved by incubating a cell lysate with the capture compound to establish a reversible binding equilibrium between the selectivity function of the capture compound and the target proteins as a second step. The mixture is irradiated by UV light within the capra box.
The irradiation photo activates the reactivity function of the capture compound. This induces the formation of an irreversible covalent cross link between the capture compound and the target proteins. Next, the capture compound protein conjugates are separated from the lysate washed thoroughly released from the solid support and tryps inized in order to identify the captured proteins by mass spectrometric analysis of the triptych peptides results are obtained that show the straightforward isolation and identification of methyltransferases from a cell lysate of esia coli based on capture compound mass spectrometry.
Hi, I'm Thomas Lentz from the Laboratory of CaTECH in the Department of Biochemistry. I'm Mathias Rega, also from the CapTech Biochemistry Laboratory. I'm Mer Linsky from the analytics department.
Today we will show you procedure for on beat capturing of methyl transferases from equalized cell ate. We use this procedure in our laboratory to study small molecule protein interactions. The procedure enables the isolation and identification of functional sub proteomes such as kinase, metalloproteinase, gtpa, ais to name a few.
We also use this approach for drug target discovery. So let's get started. For the capture experiments, the snail L homocystine caper kit short SAH caper kit is used.
This kit includes the SAH capture compound and free SAH as competitor. Also included are streptavidin coated magnetic beads with one micron diameter, five times concentrated capture buffer, and five times concentrated wash buffer using different tubes of one 200 microliter PCR tube strip prepare SAH capture compound loaded streptavidin coated magnetic beads by mixing 50 microliters of 10 milligrams per milliliter. Streptavidin coated magnetic beads with 25 microliters of 100 micromolar SAH capture compound for each Eloqua vigorously.
Shake the resulting suspensions at room temperature for two minutes to allow binding of the biotin moty of the capture compound to the streptavidin on the magnetic bead surface. Then magnetically collect the capra beads in the caps of the PCR tubes using the capra mag, discard the supernat and resuspend the resulting capra beads in 200 microliters of one times wash buffer. Again magnetically collect the capra beads in the caps of the PCR tube strips and discard the supernatant.
Then close the tubes to avoid drying of the beads. Next label new tubes for the capture assay and necessary controls, including the competition control. Pull down competition control of pull down and combined capture assay plus pull down, place the label tubes into the capra box to regulate the temperature at zero to four degrees Celsius.
Then pipette volume of milli Q water calculated for a final reaction volume of 100 microliters and 20 microliters of five times capture buffer. For each reaction, add 0.26 milligrams of a coli dh five alpha whole cell lysate to each tube and gently mixed by inversion. For the competition control and the competition control of pull down, add 20 microliters of 10 millimolar SAH competitor solution.
For the capture assay, pull down and combined capture assay plus pull down, add 20 microliters of milli Q water instead of the SAH solution, gently mix all tubes by inversion and briefly centrifuge. Then suspend the capra beads in the respective lysate and incubate for three hours at four degrees Celsius. Keeping the beads in suspension by rotation to allow reversible binding of the SAH binding proteins to the SAH selectivity function of the SAH capture compound.
Following incubation, briefly centrifuge and place the suspensions in the capra box. Separate the tubes and remove the suspensions for the pull down and competition control of pull down onto ice. Leave the remaining suspensions for the capture assay competition control and combined capture assay.
Plus pull down in the capra box to irradiate the samples with UV light while simultaneously cooling them. Irradiate the samples for a total time of 30 minutes with 2.5 minute irradiation intervals in the closed tubes between zero and four degrees Celsius. This process will form a covalent cross-link between the reactivity function of the SAH capture compound to the SAH binding proteins.
Remove the suspensions an irradiation interval of 2.5 minutes from the Capra box. Cool in ice water for about 15 seconds and mix several times by inversion. Then briefly centrifuge the samples to remove any suspension remaining in the lids and place them back into the capra box for the next irradiation interval.
Next again, include the suspensions for pull down and competition control of pull down and add 20 microliters of 10 millimolar SAH solution to the capture assay for the competition control, pull down competition control of pull down and combined capture assay plus pull down, add 20 microliters of milli Q water. Incubate the suspensions for 10 minutes at four degrees Celsius, keeping the beads and suspension by rotation in the capture assay. The SAH binding proteins not cross-linked to the SAH capture compound will be displaced.
Following incubation, collect the capra beads from the suspensions using the capra mag and discard the supernat. Then wash the beads by resus, suspending them in 200 microliters of one times wash buffer and collecting the beads by magnetic separation. Repeat this wash five times, then resuspend them in 200 microliters of milli Q water and collect the beads by magnetic separation.
Resuspend the beads in 200 microliters of 60%aceto nitrile and collect the beads using the capri mag. Repeat this acetyl nitrile wash twice. Apply ultrasound if beads cannot be readily resuspended.
Next, release the captured proteins from the beads by adding 200 microliters of freshly prepared. 60%acetyl nitrile, 0.2%Tri Fluor acetic acid followed by a 10 minute incubation at room temperature under vigorous shaking. Again, apply ultrasound if beads cannot be readily resuspended.
Finally magnetically collect the beads and discard them. Cut the tube strip into single tubes, perforate the lids and evaporate the supra natin until dry. Using a centrifugal evaporator at this stage captured proteins can either be subjected to SDS page or processed for mass spectrometry as described in the following section, dissolve the captured proteins released from the magnetic beads in 10 microliters of 50 millimolar ammonium bicarbonate.
Place the samples into an ultrasound bath, then vortex the samples. Next, add one microliter of 0.5 micrograms per microliter trypsin in one millimolar HCL briefly centrifuge and incubate the solution at 37 degrees Celsius overnight. Following overnight incubation desalt a solution containing the triptych peptides of the captured proteins using C 18 material such as 20 microliters stage tips by proxy on biosystems per manufacturer's instructions, elute the peptides twice with 10 microliter of 50%methanol, 5%formic acid.
Then evaporate the solvent using a centrifugal evaporator, leaving the dry desalted peptides dissolve the peptides in 5.5 microliter of 0.1%formic acid by applying ultrasound in an ultrasound bath and vortexing. Finally analyze the sample by nano flow liquid chromatography coupled with tandem mass spectrometry as described in the written protocol. To analyze the MSM S data, use a protein identification algorithm such as seaquest implemented in bioworks browser version 3.3 0.1, SP one and X tandem implemented in the scaffold three software.
Automated database searching is performed against the most recent universal protein resource knowledge base. Swiss Pro database released www x pai. org of the organism investigated.
The database used for the present study is a coli strain. K12 release 57 dash 11 within Seaquest set up automated database searching with a five parts per million precursor tolerance. One atomic mass unit fragment ion tolerance and full trips and specificity allowing up to two missed cleavages.
Set the variable modifications to allow for phosphorylation at seine thine and tyrosine oxidation of methionine dation at asparagines and glutamine, acetylation at lysine and sine formulation at lysine and methylation at arginine lysine sine thianine and asparagine. Do not use fixed modifications in the database search load DTA or SRF files generated by Seaquest into the Scaffold three software, which performs probability assessment of peptide assignments and protein identifications by combining seaquest and x tandem database searches. Scaffold is useful for easily comparing, validating and visualizing protein lists from several samples.
Set the parameters within the scaffold three software to accept only protein identification and to report as distinct hits. Set the hits to contain at least one discriminant peptide that passed probability validation. According to scaffold three analysis, consider only peptides with greater than or equal to 95%probability as specified by the peptide profit algorithm within the scaffold.
Three software set protein identification probabilities for multiple peptide assignments to greater than or equal to 95%according to the protein profit algorithm. For single peptide protein identifications arbitrarily set protein probability to greater than or equal to 50%and manually inspect the corresponding peptide. Ms ms spectra proteins that comprise similar peptides and cannot be differentiated based on Ms MS analysis alone are grouped by the software to satisfy the principles of parsimony.
The estimated false discovery rate of peptide identifications can be determined using the reversed protein database approach and should be less than 1%Here. SDS page, silver stain analysis of the captured intact proteins is shown in the capture assay. Excess free SAH was added after the UV irradiation step to ensure that only proteins having the capture compound covalently attached by the photo reactivity function are isolated.
Proteins were identified by MS from cut protein gel bands after the Ingel Triptych Digest. For comparison, the leftmost lane shows 0.25%of the e coli DH five alpha whole cell lysate from which proteins were isolated in the capture assay. This comparison points out the dramatic reduction of sample complexity achieved by the capture compound technology.
The controls were run in the center of the gel and include the competition control of the assay. This control contains excess free SAH as competitor before the UVI radiation step to prevent binding of proteins to the SAH capture compound via the SAH selectivity function. The competition control is essential to determine any non-specifically captured proteins that do not bind SAH.
Thus, proteins identified in the capture assay, but not in the competition control are specific. SAH binding proteins. Also included is a pull down experiment with no UV irradiation and no addition of free SAH.
The competition control of pull down is also not irradiated, but contains SAH as a competitor. Finally, the combined capture assay plus pull down is UV irradiated and no free SAH was added during the workflow. It is evident that photo cross linking enhances yield and sensitivity of the experiment and the specificity can be readily tested in competition experiments using an excess of free SAH instead of analyzing the captured intact proteins by SDS page.
The whole mixture of captured proteins can be analyzed at once by nano flow liquid chromatography coupled to tandem mass spectrometry after Triptych Digest. This is the real CCMS approach and is much more sensitive than analysis by SDS page. METHYLTRANSFERASES and other selected proteins identified by CCMS experiments are shown in this table.
The given numbers denote the unweighted peptide spectral count per protein, much more methyltransferases and other SAH binding are identified in the capture assay as compared to the pull down. And SAH specificity is shown by the almost complete absence of these proteins in the competition control. The total number of identified proteins in the CCMS runs and protein overlap between the runs was tabulated.
The number of proteins identified with at least two peptides is given in parentheses. The high reproducibility of the method can be inferred from the high protein overlap of mainly unspecific proteins between comparable experiments, especially in the proteins robustly identified with at least two peptides here. Venn diagrams describe the overlap of identified proteins in the capture assay, the competition control and the pull down while the numbers of all identified proteins are given on the right with the numbers of proteins identified with at least two peptides given in parentheses, the numbers of methyl transferases and SAH nuclease are shown to the left.
We've just shown you how to isolate and identify methyl transferases or other functional sub proteomes from e coli cell lysate or from other tissue or cell lysates by capture compound mass spectrometry or CCMS. When doing this Procedure, it is important to remember to avoid long exposure of the capture compound to visible light to keep samples cool. To freshly prepare the TFA Aceto Nitrile solution used for releasing the captured proteins from the magnetic beads and to avoid contamination of the experiments by external protein sources such as keratin originating from dust or the experimenter.
Also, we found out that it depends on the specific setup, whether the offbeat configuration, whether the photo crosslink configuration takes place between the pre recapture compound and the proteins and solution or the presently described on beat configuration performs better. So that's it. Thanks for watching and good luck with your experiments.
