Detection of protein modifications and counterfeit protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry: Studies of insulins

Hongping Ye, John Hill, John Kauffman, Connie Gryniewicz, Xianlin Han

Research output: Contribution to journalArticle

11 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)182-191
Number of pages10
JournalAnalytical Biochemistry
Issue number2
StatePublished - Aug 15 2008



  • Aggregation
  • Counterfeit
  • Deamidation
  • Generic protein drugs
  • Glu-C
  • Insulins

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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