Detection of Protein Modifications and Counterfeit Protein Pharmaceuticals Using iTRAQ and MALDI TOF/TOF Mass Spectrometry: Studies with Insulins

摘要

iTRAQ (isotope tags for relative and absolute quantification) reagent coupled with MALDI TOF/TOF mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies, and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, Lantus®) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including Human insulin (P28K29) and Lispro (K28P29), which only differ by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.