Characterization of recombinant protein mutants by top-down sequencing using quadrupole time-of-flight mass spectrometry.

Top-down sequencing using quadrupole time-of-flight mass spectrometry is used as a direct way of locating the mutated sites of recombinant proteins and posttranslational modification in a protein. Several mutants of barstar, expressed in E.coli, were confirmed by analyzing the fragmentation pattern of mutants. A contaminant protein, that appeared while purifying mutants of barstar, was identified as acyl carrier protein from E.coli with a posttranslational modification on serine residue, indicating that the protein was biologically active. A mutant of ribosomal protein S6 has been characterized with neutral loss of ammonia at the N-terminal region of the protein. The power of the "top-down" approach in characterizing the mutants of recombinant proteins has been demonstrated.