Mass spectral characterization of organophosphate-labeled lysine in peptides

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 83.9amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8amu. 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.

Figure 1

Illustration of the reaction of lysine with OP, using chlorpyrifos oxon as an example for the OP.

Figure 2

OP structures

Figure 3

CID Fragmentation of ε-N-modified lysine. The mass of R2 for DFP can be +164 for diisopropoxyphosphate, or +80 for DFP that has lost both isopropyl groups. The mass of R2 for CPO can be +136 for diethoxyphosphate, or +108 for monoethoxyphosphate, or +80 for is indicated as CPO that has lost both ethoxy groups. The immonium ion minus NH₃ immonium-NH₃.

Figure 4

A CID mass spectrum of the diisopropoxyphosphate-labeled, human serum albumin, tryptic peptide LAK*TYETTLEK. The doubly-charged parent ion at 731.0 amu is designated by the letter P. Sequential neutral loss of isopropylene (42 amu) from the diisopropoxyphosphate parent ion is designated by P1 (709.8 amu) and P2 (688.3 amu). Sequential neutral loss of isopropylene from the b3 ion is indicated by b3c (diisopropoxyphosphate form at 477.6 amu), b3b (monoisopropoxyphosphate form at 435.6 amu), and b3a (phosphate form at 363.6 amu). The values enclosed in the boxes are the masses of the characteristic fragments for diisopropoxyphosphate-labeled lysine. Lysine 351 is covalently modified by diisopropylfluorophosphate.

Figure 5

A CID mass spectrum of the diethoxyphosphate-labeled, mouse transferrin, tryptic peptide STTK*DLLFR. The values enclosed in the boxes are the masses of the characteristic fragments for diethoxyphosphate-labeled lysine. The parent ion is marked by [M+2H]⁺². Lysine 626 is labeled by chlorpyrifos oxon.

Figure 6

A CID mass spectrum of the diethoxyphosphate-labeled, bovine actin, tryptic peptide VAPEEHPTLLTEAPLNPK*ANR. The parent ion is marked by the letter P. There are no characteristic fragments for diethoxyphosphate-labeled lysine. Lysine 113 is covalently modified by chlorpyrifos oxon.

Figure 7

A CID mass spectrum of the diethoxyphosphate-labeled, bovine tubulin beta, tryptic peptide K*Y*VPR. The values enclosed in the boxes are the masses of the characteristic fragments for diethoxyphosphate-labeled lysine and diethoxyphosphate-labeled tyrosine. The parent ion is marked by [M+2H]⁺². Lysine 58 and tyrosine 59 are labeled by chlorpyrifos oxon.