TCEP-Mediated Protein Hydrolysis: Highly Selective Cleavage C-Terminal to Aspartic Acid

Tris(2-carboxyethyl)phosphine (TCEP) is a commonly used laboratory reagent for the purposes of reducing disulfide bonds. TCEP is often preferred over alternative reducing reagents, such as dithiothreitol (DTT), due to its strong redox potential, effectiveness in a broad pH range, and handling considerations. Despite this, many side reactions involving TCEP have been poorly categorized and understood.

Utilizing a combination of gel electrophoresis and mass spectrometry techniques, we have discovered a unique and novel mechanism by which TCEP engages in the hydrolysis of proteins. This behavior has been observed to be site specific to the carboxyl terminus of aspartic acid residues.

Cleavage at this location is completely independent of the reduction of disulfide bonds in proteins due to the observance of this phenomenon in proteins lacking disulfide bonds, and some lacking cysteines altogether. While the specific chemistry of this hydrolysis is unknown, this discovery has potentially wide-ranging impacts as a sample preparation tool for mass spectrometry analysis, as well as being cautionary towards other common laboratory uses of this ubiquitous reagent where it could potentially cause unwanted hydrolysis of proteins being studied.

The broader analytical implications of this finding are that many previously unknown origin artifacts can now be properly attributed to a source, particularly in areas such as proteomic sample preparation, redox chemistries studies, and similar. This also expands our knowledge of TCEP chemistry beyond simply a disulfide bond reductor, offering both methodological opportunities for peptide mapping and important cautionary implications for biochemical sample treatment.