The mass squirm protocol aims to quantify lysine demethylase activity using isotopic methylation and maldi mass spectrometry first perform enzyme catalyzed demethylation reactions on peptides containing methylated lysine. Residues then chemically methylate the peptide products of the demethylase reaction with deuterated formaldehyde to produce uniformly methylated peptides that ionize the same way by maldi proceed to quantify each peptide species with multi to mass spectrometry and account for isotopic overlap. In order to identify relative amounts of un mono dye and trimm methylated peptides, the mass squirm results determine relative amounts of each demethylase reaction product based on changes in mass.
The primary advantage of this technique over existing methods like routine moldy mass spectrometry is that mass score accounts for differences in ionization between differently methylated peptides. We first had the idea for this approach when we developed a method to measure histone acetylation levels using isotopically heavy acetic anhydride. The challenge to this technique is extracting peak area data, which can be complicated by overlap of peptide isotopic envelopes.
For the demethylase reaction combined 125 nanograms of recombinant lysine specific demethylase one enzyme with 0.25 micrograms of biotinylated dimethyl hisone H three peptide in a final volume of 20 microliters demethylase buffer. Also prepare a control sample containing 0.25 micrograms peptide without the LSD one enzyme incubate the reaction and control tubes for two hours at 37 degrees Celsius. Next to prepare R two 20 micron porous beads mix equal volumes of beads and methanol.
Then add 10 volumes of TFA formic acid, add eight microliters of porous beads to each sample and agitate for 15 minutes at room temperature. Meanwhile, condition two C 18 zip tips. Pipetting twice with 20 microliters of 0.1%TFA four times with 70%aceto nitrile 0.1%TFA, and four times again with 0.1%TFA.
Next, load the two samples onto the conditioned zip tips. With a pipetter, push the volume through the C 18 resin. Repeat until total volume of both samples has been pushed through the zip tips.
Then wash the zip tips twice with 20 microliters of 0.1%TFA elute samples in 40 microliters illusion buffer proceed to dry eent completely with a speed vac concentrator. First resuspend the sample in 100 microliters of 50 millimolar phosphate buffer pH 7.4. Then add eight microliters of bore dimethyl amine and 16 microliters of derated formaldehyde.
Incubate at four degrees Celsius for two hours. Repeat the reductive methylation by adding boring dimethyl lamine and deuterated formaldehyde and incubate for two hours. The third time, add boring dimethyl lamine and incubate at four degrees Celsius for 15 hours.
Quench the reaction with 12.5 microliters of one molar tris. pH 7.4 proceed to dry eent completely with a speed vac concentrator. First Resus, suspend the control and demethylase reaction samples in 100 microliters of 50 millimolar phosphate buffer pH 7.4.
Now add eight microliters porous beads to the control and demethylase reaction samples and collect on zip tips as shown earlier. Elude in two microliter volumes onto a MALDI sample plate and allow to dry at room temperature to crystallization. Proceed to analyze samples by mass spectrometry.
Open the spectro files in to work software and export to M over Z software. Further verify reaction products by tandem mass spectrometry. First, determine the peak area ratio of carbon 13 C two and carbon 13 C four isotopes relative to the mono isotopic peak for the control sample.
Use these calculated ratios to quantify the relative amount of peptide existing in each modification. State of the demethylase reaction sample. The LSD one enzyme can be effectively used in mass squirm quantification of lysine demethylation.
Since the control reaction does not undergo reductive methylation, it indicates the typical isotopic envelope for this peptide Resulting peak areas determine background ratios of peptide existing in carbon 13 C two and carbon 13 C four. Isotopic states, peak areas from the mass spectrum for the control reaction allow quantification of the relative amount of peptide existing in each modification state. In the demethylase reaction, the peak areas from these control and demethylase reactions determine 33.7%dimethyl lysine 4 42 0.3%monomethyl lysine four and 24%unmethylated lysine four.
This mass squam approach offers key insights into the field of histone epigenetics, such as how demethylation of histone lysine residues contributes to various diseases. While this methylation assay has been applied to study demethylases, it can also be used to study methyl transferases as well.
