Methods to Identify the NMR Resonances of the ¹³C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

The overall goal of this procedure is to assign the alpha and epsilon carbon. 13 dimethyl amine NMR resonance of the end terminal lysine, residue of lysozyme. This is accomplished by first reductively methylating lysozyme at low and high pH, with a sub geometric amount of carbon 13 formaldehyde.

The second step is to reductively methylate the low and high pH samples with excess natural abundance for formaldehyde to complete methylation. The final step is to buffer exchange the reductively methylated samples into D two O buffer for NMR analysis. In the second method, the goal is achieved by amino peptidase degradation of a lysozyme sample.

The second step is to reductively methylate the truncated protein sample with excess carbon 13 formaldehyde, followed by buffer exchange for NMR analysis. Ultimately, NMR spectroscopy is used to show the absence of carbon 13 methyl resonance to make assignments. The main advantage of this technique over existing methods is that it allows for the unambiguous assignment of both the alpha and SSON dimethyl lamine and ML resonances of the internal lysine of a protein Label three one milliliter micro centrifuge tubes with F-L-S-L-P-H and HPH dissolve three samples of lysozyme in previously prepared buffer solution at pH's six, 7.5, and 10 to give a final concentration of five milligrams per milliliter.

Cover the tubes with aluminum foil to protect them from degradation due to light exposure. Following this add 6.1 microliters of a previously prepared one molar dimethyl amine boring complex solution to the FLS tube and 3.2 microliters of the same solution to the LPH and HPH tubes. After gently vortexing the mixtures add 12.3 microliters of a previously prepared one molar carbon 13 formaldehyde solution to the FLS sample and 6.1 microliters of the same solution to the L-P-H-N-H-P-H samples.

Shake the reaction mixtures at four degrees Celsius for two hours. Repeat the previous steps by adding the same aliquots of DMAB and carbon 13 formaldehyde to the corresponding tubes. Shake the reaction mixtures overnight at four degrees Celsius for a total reaction time of 18 to 24 hours after shaking exchange each sample's buffer with three milliliters of phosphate buffer at pH 7.5 to remove excess reagents and side products using a centrifugal filter at a three kilodalton molecular weight cut off, concentrate the samples to 500 microliters using a centrifuge with a 35 degree fixed angle rotor at four degrees Celsius and 7, 500 RCF.

Repeat the previous steps two times following this. Cover the partially labeled sample tubes with aluminum foil and add 6.1 microliters of a freshly prepared one molar DMAB solution to them. After the samples have been vortexed, add 12.3 microliters of a freshly prepared one molar natural abundance formaldehyde solution to the tubes.

Shake the reaction mixtures at four degrees Celsius for two hours. Add the same aliquots of DMAB and natural abundance formaldehyde as in the previous step, and shake the reaction mixtures overnight at four degrees Celsius for a total reaction time of 18 to 24 hours following buffer exchange with 50 millimolar sodium phosphate buffer. Transfer 10 microliter aliquots of each sample to clean micro centrifuge tubes and store the samples at minus 20 degrees Celsius for MALDI MS analysis.

Next, add 375 microliters of a previously prepared two molar sodium borate buffer solution to four 15 milliliter conical tubes. Para film the tops of the tubes and poke holes through the para film. Place the tubes in a lyophilized flask and freeze the sample.

Once the samples have been frozen, place a flask on a lyophilizer to dry after removing the sample from the lyophilizer pipette 15 milliliters of D two O to each tube and mix following this label, three four milliliter conical tubes containing centrifugal filters with F-L-S-L-P-H and HPH. Add approximately 500 microliters of each sample to the corresponding filter tubes. Then add three milliliters of 50 millimolar sodium ate buffer.

Add pH 8.5 to each tube. Concentrate the samples to 500 microliters using a centrifuge with a 35 degree fixed angle rotor at four degrees Celsius and 7, 500 RCF. After emptying the trap under each filter, add another three milliliters of buffer to the samples and concentrate them to 500 microliters.

Repeat the previous steps four times using the bissy acid protein assay. Determine the concentration of each sample. Dilute the lysozyme samples with 50 millimolar sodium void buffer to give a final concentration of 150 micromolar.

Next, transfer 530 microliters of each sample to a five millimeter NMR tube. Then prepare an 80 millimolar stock solution of carbon 13 labeled one two DI CHLORO ethane in D two O.Dilute the solution to 24 millimolars with D two O and then transfer it to a coaxial insert tube to use as an NMR reference. After running A 1D proton carbon H-S-Q-C-N-M-R integrate the peak areas for each resonance.

Prepare a five milligram per milliliter solution of lysozyme in ultrapure water. Transfer 250 microliters of the lysozyme solution to a clean one milliliter centrifuge tube. Then add 25 microliters of a 0.1 molar trice solution at pH eight and six microliters of a two point 16 microgram per milliliter.

A romanis proteolytic amino peptidase solution to the centrifuge tube. Incubate the mixture at 37 degrees Celsius for six hours. After incubation, add an additional six microliters of the amino peptidase solution to the sample to give a one to 50 molar ratio of amino peptidase to incubate for an additional 20 hours at 37 degrees Celsius.

Following this DESALT 30 microliters of the sample using a C 18 spin column and elute the sample in 80%ACEDONITE trial per 0.1%TFA store the sample at minus 20 degrees Celsius. For maldi MS analysis, prepare the remaining protein sample for NMR as described previously, A pH induced chemo selective reductive carbon 13 methylation reaction has been demonstrated on lysozyme at low pH. The reaction prefers the end terminal alpha amine over the side chain epsilon amine and vice versa at high pH as illustrated in the NMR spectra here.

Peak seven is absent for the lysozyme sample reacted at pH 10 indicating that peaks one to six belong to epsilon dimethyl amino groups and peak seven is the end terminal alpha dimethyl amino group. Alpha Amin favored reductive carbon. 13 methylation at pH six was observed when the amount of carbon 13 formaldehyde used was reduced to a molar ratio of two to seven, and the sample was not further reacted with natural abundance formaldehyde as illustrated here, the methylation at low pH is a mixture of monomethyl amines and dimethyl amines.

Three amino groups show carbon 13 dim methylation indicating that these groups reacted faster than the groups showing carbon. 13 monomethyl of the three carbon 13 dimethyl amines. Peak seven has the highest intensity and was assigned as the N termina alpha dimethyl amine group corroborating the assignment of peak seven.

Using the sample reacted at high pH with the ability to alter the rate of reductive methylation of the N terminal alpha amino group. The MS assisted method for assigning the NMR resident can be applied. The lysozyme sample that was reductively carbon 13 methylated at pH 10 was digested with trypsin and analyzed with maldi TOF MS because the alpha amino group was not reductively carbon 13 methylated.

Under these conditions, the MS isotopic profile of the tetra methylated N terminal peptide can be used to quantify the percentage of carbon 13 incorporated at the N terminal epsilon dimethyl amino group. The carbon 13 labeled DIM methylated N terminal peptide 6 6 2 gave a scaled percentage of carbon. 13 incorporation of 27%NMR analysis of the same lysozyme sample gave a percentage of carbon 13 incorporation of 33%for peak six, indicating that peak six is the end terminal lysine epsilon carbon 13 dimethyl amine Maldi, TOF mass spectra of untreated and amino peptidase treated lysozyme samples are pictured here.

Insulin, which was used as a positive control, shows a shift to lower average master charge ratio compared to its native form of 5731.2. The treated lysozyme shows a shift from 14305.8 to 13930.5, corresponding to the loss of the first three end terminal residues of lysozyme, lysine, valine, and phenylalanine. Shown here are the NMR spectra of the untreated and amino peptidase treated reductively carbon 13 methylated lysozyme samples compared to the controlled spectrum, the spectrum of the amino peptidase treated sample shows a decrease in intensity for peaks six and seven and an additional peak labeled seven A From this data peaks six and seven can be assigned pairwise as lysine, alpha and epsilon dimethyl amine.

Before attempting this procedure, it's important to consider the solubility of your protein, the various phs, and adjust the protocol accordingly.