Elsevier

Analytical Biochemistry

Volume 394, Issue 1, 1 November 2009, Pages 92-100
Analytical Biochemistry

Mass spectral characterization of organophosphate-labeled lysine in peptides

https://doi.org/10.1016/j.ab.2009.07.008Get rights and content

Abstract

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9 amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8 amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.

Section snippets

Materials

Purified human serum albumin (essentially fatty acid free, cat. no. 05418), bovine actin (cat. no. 3653), human epidermal keratin (cat. no. K0253), DFP (cat. no. D0879), iodoacetamide (cat. no. I6125), Glu-fibrinopeptide B (cat. no. F3261), and mouse transferrin (cat. no. T0523) were obtained from Sigma–Aldrich–Fluka (St. Louis, MO, USA). Dithiothreitol (electrophoresis grade, cat. no. BP172-25) was obtained from Fisher Biotech (Fair Lawn, NJ, USA). Modified porcine trypsin (N-tosyl-l

OP-labeled peptides

A lysine from each of 22 peptides was found to have reacted with either CPO or DFP. Those 22 peptides came from seven different proteins: 5 peptides from human serum albumin, 2 peptides from human keratin 1, 1 peptide from human keratin 10, 6 peptides from bovine tubulin alpha, 1 peptide from bovine tubulin beta, 5 peptides from bovine actin, and 2 peptides from mouse transferrin (see Table 1).

When CPO reacts with lysine, the mass added to the peptide is 136 amu (for diethoxyphosphate). When DFP

Discussion

Reaction of OPs with tyrosine in a variety of proteins has been thoroughly documented [31]. We have now found a comparable reaction between OPs and lysine. Table 1 lists 22 peptides from seven proteins that are labeled on lysine. Involvement of this many different proteins strongly suggests that the reaction is commonplace. Nonetheless, we are aware of no other reports of OPs reacting with lysine on protein.

Analysis of the MS/MS spectra for 4 of the 22 peptides was presented. Comparable data

Acknowledgments

This work was supported by the U.S. Army Medical Research and Materiel Command (W81XWH-07-2-0034 to O.L.), the National Institutes of Health (U01 NS058056 to O.L. and P30CA36727 to Eppley Cancer Center), the Direction Générale de l’Armement of the French Ministry of Defense (DGA 03co010-05/PEA01 08 7 and DGA/PEA 08co501), and the Agence Nationale pour la Recherche (ANR-06-BLAN-0163). Mass spectra were obtained with the support of the Mass Spectrometry and Proteomics Core Facility at the

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