P142 Untargeted analysis of protein modification by drug-related chemicals using background subtraction and proteomic approach

Ming Yao , Biotransformation, Bristol-Myers Squibb Co, Princeton, NJ
Haiying Zhang , Dept of Biotransformation/HW21-1-15, Bristol-Myers Squibb Co, Princeton, NJ
Jinping Gan , Department of Biotransformation-PCO, Bristol-Myers Squibb, Princeton, NJ
William G. Humphreys , Department of Biotransformation-PCO, Bristol-Myers Squibb, Princeton, NJ
Covalent modification of cellular proteins by chemically reactive compounds/metabolites has the potential to disrupt biological function and elicit serious adverse drug reactions. Information on the nature and binding patterns of protein targets are critical toward understanding the mechanism of drug induced toxicity. Since measurement of total protein covalent binding provides little information on the nature of the modified proteins, a shotgun proteomics approach was reported 1 recently for characterization of the human liver microsomal (HLM) subproteome, with a known reactive metabolite 2-(methylsulfonyl)benzo[d]thiazole, and its parent compound, (methylthio)benzo[d]thiazole. Numerous modified peptides have been identified based on the anticipated target modification in proteomic database search. An alternative to this targeted approach is the use of untargeted background subtraction algorithm. The objective of this study is to reanalyze the same dataset from that study to see if untargeted background subtraction approach have the detection sensitivity and selectivity similar to targeted proteomic approach. First of all, the dataset generated by LC/MS (Thermo Orbitrap) was sliced and extracted to get full scan MS dataset and then converted it to ANDI format, then subtracted from control by in-house software. In addition, the same dataset was analyzed by a proteomic software, PEAKS, to search for the modified peptides Unidentified peaks found by background subtraction was further analyzed through De Nova sequencing in Peaks software. Result demonstrated over 20 modified peptides has been identified based on background subtraction and proteomic approach, which was very similar with those results reported previously. Therefore it indicated the detection and identification of HLM proteins modified by a reactive metabolite can be accomplished by both targeted proteomic approach and untargeted background subtraction. The key limitation of the proteomic approach is the requirements of known structures of protein modifications. The untargeted background subtraction approach can reach the same levels of detection sensitivity and selectivity as the proteomic approach although additional confirmation is needed. The combination of the background subtraction approach with the proteomic approach is a powerful tool for identification of known and unknown protein adducts.

Reference:

1. Yanou Yang et al, Chem. Res. Toxicol., 2014, 27 (9), pp 1537–1546