Amino Acids, Peptides and Proteins - Devenyi T., Gergely J. 1976

Methods of medium- and high-voltage electrophoresis
Diagonal electrophoresis method
Isolation of lysine-containing peptides

Analysis of Lysine-containing Peptides from enzymatic (but not tryptic!) or partial chemical hydrolysates is an important step in deciphering the Amino Acid Sequence of Proteins. Isolation of lysine-containing peptides should begin with the reversible blocking of the ε-amino groups of lysine residues in the target protein. Such reversible modification can be achieved by trifluoroacetylation [5], maleylation [2], or citraconylation [4].

Prior to trifluoroacetylation, the SH groups of the target protein are carboxymethylated or oxidized with performic acid. 100 mg of protein is dissolved in 10 mL of 8 M urea solution, and 3 mL of ethyl thioltrifluoroacetate is added to this solution in three portions with thorough stirring, maintaining the pH at approximately 10 using 5 N NaOH solution. After adding the final portion of ethyl thioltrifluoroacetate, the pH is lowered to 6, and urea and excess reagent are removed by dialyzing the mixture against distilled Water or a 1% ammonium bicarbonate solution. As a result of trifluoroacetylation, the ε-amino groups of lysine residues are blocked by trifluoroacetyl groups, preventing their protonation. Next, the modified protein is subjected to Enzymatic Hydrolysis, ensuring that the pH does not exceed 8.2, as trifluoroacetyl groups are cleaved in more alkaline environments. The hydrolysate is subjected to electrophoretic Separation, then a strip is cut from the electrophoregram for analytical diagonal Electrophoresis and placed for 4 h in a desiccator over 5 N ammonia solution. In an alkaline medium, the trifluoroacetyl groups are cleaved and the ε-Amino groups are regenerated, becoming available for protonation once again. As a result of protonation, the charge of each lysine residue changes by +1 unit. Consequently, during electrophoresis, the lysine-containing peptides shift from the diagonal position of the other components toward the cathode [10].

Reversible blocking of ε-amino groups can also be achieved through maleylation or citraconylation reactions. These reactions involve the incorporation of a carboxyl group into the ε-amino group, thereby altering its charge.

For maleylation, the target protein is dissolved in 8 M urea containing 0.1 M pyrophosphate to a concentration of 10 mg/mL. One drop of phenolphthalein is added to this solution, and 30 molar equivalents of pure maleic anhydride (calculated based on the number of amino groups) are added in three portions with thorough stirring. By continuously adding 10% NaOH solution, the pH of this mixture is constantly maintained within the color transition range of phenolphthalein. Upon completion of the reaction (after 10–12 min), urea is removed by dialysis against distilled water or by Gel filtration on a Sephadex G-25 (coarse) Column.

The citraconylation reaction is carried out in the same manner as the maleylation reaction, in an 8 M urea solution containing 10 mg/mL of protein, using an autotitrator. The pH value is maintained at a constant level by continuous addition of 10% NaOH solution. To the protein solution, 30 molar equivalents of citraconic anhydride (calculated based on the number of amino groups) are added in three portions with thorough stirring. At the end of the reaction, the mixture is desalinated by dialysis or gel filtration.

Almost all Proteolytic Enzymes can be used for the enzymatic hydrolysis of maleylated and citraconylated proteins, with the exception of Pepsin, which has its optimum activity at acidic pH. Partial Acid Hydrolysis is also unsuitable in this case. After enzymatic hydrolysis, Preparative Electrophoresis is performed, then a strip is cut for analytical diagonal electrophoresis and placed in a vacuum desiccator. If a maleylated protein is being analyzed, the desiccator contains a buffer solution consisting of 1% pyridine and 5% acetic acid; a vacuum is created in the desiccator, and it is placed in an incubator at 60°C for 16 h. If a citraconylated protein is being analyzed, the desiccator contains 0.05 M acetic acid, and after creating a vacuum, it is placed in an incubator at 37°C for 6 h. After incubation, the strips are dried, sewn onto new sheets of filter paper, and subjected to electrophoresis in a direction perpendicular to the original. Upon Cleavage of the acyl groups, the amino groups, which carry a +1 charge, are regenerated. Since the blocking carboxyl group carried a -1 charge, the removal of the blocking agent results in a net charge increase of +2 units. This charge change leads to a highly significant shift of the lysine-containing peptides toward the cathode [2, 4].