FP-biotin

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Grigoryan, H., Schopfer, L.M., Thompson, C.M., Terry, A.V., Masson, P., Lockridge, O. (2008). Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin a potential mechanism of long term toxicity by organophos-phoms agents. Chem. Biol. Interact. April 22. (Epub ahead of print)... 

Miee treated with a nontoxie dose of a biotin-tagged OP called FP-biotin had FP-biotinylated albumin in blood and... 

In a 1963 Pedler lecture, Sanger reported that H-DFP makes a covalent bond with tyrosine in the sequence Arg-TjrThrLys from human and rabbit albumin (Sanger, 1963). Mass spectrometry of human albumin treated with soman, chlorpyrifos oxon, FP-biotin, diehlorvos, and DFP confirmed OP modification on Tyr 411 in peptide LVRY TKKVPQVSTPTL (Li et al, 2007, 2008a), where the underline shows Sanger s sequence and the indieates the labeled tyrosine. 

We soon learned differently. In a general seareh for proteins that bind OP, we treated live mice and mouse tissues with FP-biotin, a modified OP designed for identifying unknown targets of OP reaction. The FP-biotinylated... 

During years of mass spectrometry analysis, we had consistently identified OP-labeled tubulin in mouse brain. Therefore, we studied pure bovine tubulin (Cytoskeleton, Inc.) by treating it with soman, sarin, FP-biotin, DFP, chlorpyrifos oxon, and dichlorvos. We isolated the OP-labeled tryptic peptides and analyzed them by fragmentation in the QTRAP 2000 and QTRAP 4000 mass spectrometers. We identified five OP-labeled peptides in tubulin (Grigoryan et al, 2008). In every peptide, the OP was covalently attached to tyrosine (Table 56.1). 

Similar mass spectrometry experiments with pure human and mouse transferrin (Li et al., 2008c), and with human kinesin showed that the OP label was consistently on tyrosine (Table 56.1). Studies with human plasma identified OP labeling on tyrosine in apolipoprotein and alpha-2-glyco-protein. Aggressive treatment of hiunan albumin with FP-biotin and chlorpyrifos oxon led to identification of seven OP-labeled tyrosines (Ding et al, 2008). Finally, we found that synthetic peptides made a covalent bond with DFP, chlorpyrifos oxon, and dichlorvos (Table 56.1). Mass spectrometry conclusively proved that the OP was attached to tyrosine. 

The biotinylated OP called FP-biotin, when given to mice at a dose that caused no cholinergic signs of toxicity, labeled at least 12 proteins in mouse plasma. Four of the.se proteins have been identified as AChE, BuChE, carboxylcstcrasc, and albumin (Peeples et al., 2005). Thi.s experiment demonstrates that this particular OP binds to at least 12 proteins. 

Butyrylcholincsterase in plasma Pesticides, nerve agents, DFP, echothiophace. FP-biotin Aclivity assay Mazur and Bodansky (1946), Casida and (Juislad (2004), Peeples et al. (2005)... 

Schopfer, L.M., Champion, M.M., Tamblyn, N., et al., 2005. Characteristic mass spectral fragments of the organophosphorus agent FP-biotin and FP-biotinylated peptides from trypsin and bovine albumin (Tyr410). Anal. Biochem. 345, 122-132. 

Ding, S.J., Carr, J., Carlson, J.E., et al., 2008. Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin. Chem. Res. Toxicol. 21 (9), 1787-1794. 

Mice treated with a nontoxic dose of a biotin-tagged OP called fluorophosphate (FP)-biotin had FP-biotinylated albumin in blood and muscle (Peeples et al., 2005). In vitro experiments identified the site in human albumin for covalent attachment of a variety of OPs as tyrosine 411 (Li et al., 2007). 

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