Journal
ANALYTICAL BIOCHEMISTRY
Volume 379, Issue 2, Pages 182-191Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2008.04.037
Keywords
iTRAQ/MALDI; insulins; Glu-C; deamidation; aggregation; generic protein drugs; counterfeit
Funding
- NIA NIH HHS [R01 AG031675] Funding Source: Medline
- NIDDK NIH HHS [P30 DK056341-08, P30 DK056341-07] Funding Source: Medline
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Isotope tags for relative and absolute quantification (iTRAQ) reagent coupled with matrix-assisted laser desorption/ionization tandem time-of-flight (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, and 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 insulin (K28P29), which differ only 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. Published by Elsevier Inc.
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