Diagnostic Utility of N-terminal TMPP labels for Unambiguous Identification of Clipped Sites in Therapeutic Proteins

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphoniumbromide (TMPP) followed by proteolysis and mass spectrometric analysis.  Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at 273 Da, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC-MS method for the facile generation of the TMPP reporter ion at m/z 533 Da and, in some instances 590 Da, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMMP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operator Characteristic’s (ROC) of TMPP reporter ions of m/z 533 Da and 590 Da. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.