Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry

A de novo sequencing program for proteins is described that uses tandem MS data from electron capture dissociation and collisionally activated dissoci ation of electrosprayed protein ions. Computer automation is used to conver t the fragment ion mass values derived from these spectra into the most pro bable protein sequence, without distinguishing Leu/Ile. Minimum human input is necessary for the data reduction and interpretation. No extra chemistry is necessary to distinguish N- and C-terminal fragments in the mass spectr a, as this is determined from the electron capture dissociation data. With parts-per-million mass accuracy (now available by using higher field Fourie r transform MS instruments), the complete sequences of ubiquitin (8.6 kDa) and melittin (2.8 kDa) were predicted correctly by the program. The data av ailable also provided 91% of the cytochrome c (12.4 kDa) sequence (essentia lly complete except for the tandem MS-resistant region K-13-V-20 that conta ins the cyclic heme). Uncorrected mass values from a 6-T instrument still g ave 86% of the sequence for ubiquitin, except for distinguishing Gln/Lys. E xtensive sequencing of larger proteins should be possible by applying the a lgorithm to pieces of approximate to 10-kDa size, such as products of limit ed proteolysis.