Practical Protein Chemistry - A. Darbre 1989

Application of electron impact mass spectrometry for determining the amino acid sequence of peptides and proteins
Sample preparation methods
Permethylation

Acetylpeptides are permethylated as follows. First, the required base, methylsulfinyl carbanion, is prepared. For this purpose, sodium hydride (two full microspatulas) is added to 3–4 ml of dimethyl sulfoxide, and the reaction mixture is kept at 90 °C for 20 min. The progress of the reaction is judged by the color intensity of the solution and The rate of hydrogen bubble evolution. If the reaction mixture turns orange-brown within this time and hydrogen evolution virtually ceases, the reaction is considered to be complete. If hydrogen evolution stops before the desired color appears, another microspatula of sodium hydride should be added to the reaction mixture, and the reaction is continued until the mixture acquires the proper color. If the solution turns green or red during the reaction, it should be discarded. Upon completion of the reaction, the mixture is cooled and centrifuged for 10 min at 3,000 rpm. As a result, a light honey-colored liquid containing the base required for further use collects in the upper part of the centrifuge tube.

To the acetylpeptide dissolved in a drop of dimethyl sulfoxide, 20–30 drops of the solution containing methylsulfinyl carbanion are added. To ensure that the base has been added in excess, a drop of the reaction mixture is placed on a triphenylmethane crystal, which turns a deep red color in this case. Approximately one minute after adding the excess base, 500 ml of methyl iodide is added to the reaction mixture, and methylation is carried out for 70 s [21, 22]. After the specified time, the reaction is quenched by adding 2 ml of Water. The permethylated acetylpeptides are immediately extracted with 1 ml of chloroform, which has been previously washed twice with 2 ml of water, and the chloroform extract is evaporated under a stream of nitrogen. To completely remove traces of water and chloroform, the product is dried in vacuo.