T-3-sequencing: Targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry

A novel extension of the top-down approach is introduced for the selective characterization of protein termini that does not involve proteolytic digestion steps. N- and C-terminal peptides were generated from intact proteins in the mass spectrometer and further analyzed by MS/MS-an approach referred to as T-3-sequencing. N-terminal and C-terminal fragment ion series were obtained by the pseudo-MS/MS technique in-source decay (ISD) on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). These ions provided near-terminal sequence tags from the undigested protein in the ISD spectrum acquired in reflector mode and allowed to screen for the proper processing state of the terminus with respect to a reference sequence. In the second step of T-3-sequencing, the precursor ions, which have been generated by ISD and which included the Nor C-terminal sequence, were selected in the timed ion gate of a MALDI-TOF/TOF mass spectrometer for MS/MS analysis. These spectra allowed identification of the protein, the proper definition of both termini, and allowed confirmation of suspected terminal modifications. T-3-Sequencing appears to be an alternative to classical Edman sequencing, which is fast and even permits the analysis of N-terminally blocked proteins and their C-terminus.