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.
Deepalakshmi
Putchen Dakshinamoorthy
PD
Mass Spectrometry Facility, National Centre for Biological Sciences - TIFR, GKVK PO, Bellary Road, Bangalore-560 065, India. pddeepalakshmi@gmail.com
D001426:Bacterial Proteins / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics*; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C057438:barstar protein, Bacillus amyloliquefaciens