Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

The overall goal of the following experiment is to investigate the substrate specificity of protein lysine methyl transferases or PK mts. This is achieved by identification of an initial substrate and preparation of peptide arrays with several variants of this substrate containing single amino acid exchanges at different positions. As a second step, the peptide array is incubated with the PKMT in the presence of a radioactive methyl group donor, which allows detection of peptide methylation by transfer of radioactivity to the corresponding peptide spot.

Next, the array is exposed to an x-ray film and quantitatively analyzed in order to determine the methylation activity at each of the peptides. And to define the specificity profile of the PKMT, the results show which amino acids in the substrate peptide are critical for its methylation by the PKMT. The main advantage of this technique or existing methods like methylation of individual peptides is that a number of different peptides can be tested in one experiment in a cost efficient manner.

This method can help answer key question in the protein lym methylation field, such as specification of previously unknown novel substrates of PK mtss. So this method can provide insight into the specificity of PK mts. It can also be applied to other systems such as protein kinases, other protein method, transferases, or add to transferases.

I first had the idea for this method when I became familiar with the possibility of preparing large custom peptide arrays at reasonable costs using peptide spot synthesis To begin this procedure, design peptide sequences for the synthesis using the multi pep spotter software. Enter the peptide sequences as a single letter code with each letter defining the specific amino acid following this pre swell, the amino peg functionalized spot membrane in end, end dimethylformamide or DMF for 10 minutes. When finished, place the wet membrane on the spot synthesizer frame without any air bubbles or wrinkles.

Dry the membrane completely for at least 10 minutes before starting the synthesis program. Wash the membrane three times with 100%ethanol to generate the amide bond With the free amino group on the membrane. Activate the carboxy group of the incoming amino acid by adding DIC and ethyl hydroxy amino sino acetate to the amino acid derivative.

Following activation, spot the desired volumes of activated amino acids on the amino peg functionalized membrane using the programmable robot. Repeat the previous step three times to ensure better coupling of the first activated amino acid. After incubating the membrane for 20 minutes, wash it with DMF to remove uncoupled amino acids after drying and blocking the free amino groups.

Wash the membrane with DMF four times and then incubate with 20%piperine in DMF for 20 minutes to remove the FM protecting group. After washing and drying the membrane, repeat the spotting and F MOC removal steps until the desired peptide length is achieved. Following the last F MOC group D protection, wash the membrane with DMF and 100%ethanol.

Then treat the membrane with 0.02%brom phenol blue in DMF for a minimum of five minutes until it completely turns blue. After washing and drying the membrane, remove it from the synthesizer and place it in a chemical resistant box. Treat it with 20 to 25 milliliters of a side chain D protection mixture, consisting of 95%trichloroacetic acid to cleave the side chain protecting groups and scavenger reagents to protect the amino acid side chains from modification.

After ensuring that the deep protection mixture covers the membrane, completely close the box and incubate it for one to two hours while shaking gently following incubation. Wash the membrane six times with 20 to 25 milliliters of di chloro methane for two minutes each. After washing twice with 100%ethanol, dry the membrane in a desiccate overnight.

Next, pre incubate the peptide array membrane in a sealed plastic bag containing the respective methylation buffer without enzyme and amet with radioactively labeled methyl group For 10 minutes, discard the pre incubation buffer and incubate the membrane in eight milliliters of methylation buffer containing the respective PKMT for one to two hours. Following this, discard the methylation buffer in a radioactive waste container and wash the peptide arrays five times with 20 milliliters of buffer containing 100 millimolar ammonium bicarbonate, and 1%STS for five minutes to remove the bound protein. After discarding the last washing buffer in the radioactive waste container, incubate the membrane for five minutes in 10 milliliters of amplify solution.

Then discard the amplify solution in the radioactive waste container for organic solvents. Seal the peptide array in a plastic bag and put it in an auto radiography cassette. Place the auto radiography film on the peptide in a dark room.

Then close the cassette carefully. After exposing the film at minus 80 degrees Celsius, develop it to analyze the results. Finally, capture the image a couple of times with different exposition times to avoid saturation of strongly methylated peptide substrates.

An example of a methylation reaction of a peptide array with NSD one is shown here. The horizontal axis represents the sequence of the peptide and in the vertical direction. The altered amino acid is indicated methylation with nsd, one shows that its specifically acts on H three K 36 and recognizes three to four amino acid residues on either side of the target lysine consolidation of the three peptide array experiments of NSD one is shown here.

Around 85%of the peptides show standard deviations smaller than plus or minus 20%and more than 97%of the peptides demonstrate standard deviations smaller than plus or minus 30%The calculated discrimination factor data shows that NSD one prefers aromatic residues at the minus two position. At the minus one and plus two sites, it prefers hydrophobic atic residues. And at the plus one site, there is a preference for arginine, lysine, glutamine, or asparagine.

Potential novel peptide substrates can be found by database searches of the human proteome. Strong methylation of some of these peptides with ome confirm their identity as novel NSD one peptide substrates. While attempting this procedure, it's important to remember to use the most appropriate initial substrate Following this procedure.

Other methods like methylation of the correspondent proteins in vitro and in cells can be performed in order to find out if proteins are accepted as substrates, as well After its development. This technique paved the way for researchers in the field of protein lysine mesylation, to define the substrate specificity of many PK mts and identify several novel substrates. This helped to understand the important biological function of PK mts.

Don't forget that working with radioactivity and organic solvents in peptide synthesis can be assert, and appropriate precautions should always be taken while performing this procedure.