Characterization of Recombinant Protein Mutants by Top-down Sequencing Using Quadrupole Time-of-Flight Mass Spectrometry

Abstract

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 post-translational 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 an acyl carrier protein from E. coli with a post-translational 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.