Abstract

Counting needles in a haystack: improving sensitivity and quantitation of low-level tryptic peptides, Online AB Sciex Mass Spectator Newsletter, Volume 5, March 2014, pp 8-11.

Carmen Fernández-Metzler, president of PharmaCadence Analytical Services, believes that quantitation should not be the limiting factor in biological studies. She took this mantra to heart when determining the basal levels of the membrane-bound isoforms of UDP-glucuronsyl-transferase (UGT), a major enzyme in the phase II-elimination of over 200 xenobiotic drugs and endogenous metabolites. Having a good understanding of UGT’s role in drug metabolism, by correlating both the activity and absolute protein levels, provides a handle on effective dosages for clinical trials. But, these calculations require exact protein quantitation at very low cellular concentrations—around 2-100 pmol/mg of microsomal tissue.
When tackling this issue with the UGT family of enzymes, Dr. Fernández Metzler was presented with a challenging situation. \“We didn’t have a pure UGT protein standard at the time, so the difficulty was in quantitation of overexpressed recombinant protein. Additionally, peptide concentrations did not always agree with each other due to variable digestion efficiency and recovery,\” explained Dr. Fernández-Metzler. To address these problems, a strategic workflow was devised: isotopic dilution of signature peptides, tightly-controlled tryptic digestions, and analysis using the sensitive AB SCIEX QTRAP® 6500 System and highly-reproducible chromatographic separations using the Eksigent microLC System to achieve quantitation of six endogenous UGT isoforms in a complex matrix

 

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