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1.
FIG. 6.

FIG. 6. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

OP-labeled Tyr 238 and Tyr 574 in the crystal structure of human apo-transferrin (PDB code 2hav). The phenolic hydroxyl of Tyr 238 is 3.37 Å from the amine of Lys 239 and 5.06 Å from Gln 92. The phenolic hydroxyl of Tyr 574 is 4.06 Å from the imidazole nitrogen of His 535 and 5.99 Å from oxygen in the carbonyl of Glu 572 (Wally et al., 2006).

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
2.
FIG. 7.

FIG. 7. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

Labeling of peptide ArgTyrThrArg by dichlorvos, chlorpyrifos oxon, and DFP. The reaction of 170μM peptide with 0.1, 0.2, or 1mM OP was in ammonium bicarbonate buffer pH 8.3, at 37°C for 16 h. MALDI-TOF cluster areas were used to calculate % labeling. MS/MS spectra identified the labeled residue as tyrosine.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
3.
FIG. 8.

FIG. 8. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

FP-biotinylation of Tyr 238 and Tyr 574 in human transferrin does not interfere with ferric ion binding. One milliliter of 12.1μM apo-transferrin control (♦) titrated with FENTA had an endpoint of 27.3μM FENTA, which calculates to 2.26 mol of ferric ion bound per mole of transferrin. One milliliter of 14.67 μM FP-biotinylated apo-transferrin (▪) had an endpoint of 31.3μM FENTA, which calculates to 2.11 mol of ferric ion bound per mole of transferrin. Assays for each type of transferrin were performed in duplicate.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
4.
FIG. 2.

FIG. 2. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

Covalent binding of FP-biotin to human alpha-1-antitrypsin, human complement C3, human alpha-2-macroglobulin, and bovine holo-transferrin. (A) Coomassie stained SDS gel. (B) Blot hybridized with Streptavidin Alexa Fluor 680 to visualize FP-biotinylated proteins transferred to PVDF membrane from the gel. Lane 1, FP-biotinylated alpha-1-antrypsin; lane 2, unlabeled alpha-1-antitrypsin; lane 3, FP-biotinylated complement C3; lane 4, unlabeled complement C3; lane 5, FP-biotinylated alpha-2-macroglobulin; lane 6, unlabeled alpha-2-macroglobulin; lane 7, FP-biotinylated transferrin; lane 8, unlabeled transferrin.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
5.
FIG. 3.

FIG. 3. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

Structures of the phosphoryl moieties that have become attached through phosphorus to tyrosine. Covalent binding to tyrosine results in loss of the fluoride ion from soman, DFP, FP-biotin, and sarin, of the dichlorovinyl alcohol group from dichlorvos, and of the trichloropyridinol group from chlorpyrifos oxon. Tyrosine loses one hydrogen. The added mass is 162.2 for soman, 164.1 for DFP, 136.0 for chlorpyrifos oxon, 572.3 for FP-biotin, 108.0 for dichlorvos, and 120.0 for sarin. The arrows in FP-biotin indicate fragmentation sites. A 227 amu ion is produced by cleavage between carbon 16 and the adjacent nitrogen. A 329 amu ion is produced by cleavage between carbon 10 and the adjacent nitrogen. The 312 amu ion is produced by loss of the amine from the 329 ion.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
6.
FIG. 1.

FIG. 1. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

Blots showing FP-biotin–reactive proteins in human (A) and mouse (B) plasma. FP-biotinylated plasma proteins were separated on nondenaturing polyacrylamide gels and transferred to PVDF membranes. Blots were hybridized with the fluorescent probe Streptavidin Alexa Fluor 680. A1, 20 μg human transferrin; A2, blank; A3, 20 μg FP-biotinylated human transferrin; A4, blank; A5, 5 μl of human plasma; A6, blank; A7, 3.3 μl of FP-biotinylated human plasma; A8, 6.6 μl of FP-biotinylated human plasma; A9, 9.9 μl of FP-biotinylated human plasma; A10, blank; A11, 1 pmol FP-biotinylated human butyrylcholinesterase (BChE). B1, 20 μg mouse transferrin; B2, blank; B3, 20 μg FP-biotinylated mouse transferrin; B4, blank; B5, 5 μl of mouse plasma; B6, blank; B7, 5 μl of FP-biotinylated mouse plasma; B8, 10 μl of FP-biotinylated mouse plasma; B9, 15 μl of FP-biotinylated mouse plasma. The heavy band in B contains mouse ES1 carboxylesterase and mouse albumin. ES1 carboxylesterase in mouse plasma does not separate well from albumin on a nondenaturing gel. Human plasma does not contain carboxylesterase.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
7.
FIG. 4.

FIG. 4. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

MS/MS spectra of OP labeled Tyr 238 in peptide KPVDEYK of human transferrin. Mass spectra in (A-D) were acquired on the QTRAP 4000 mass spectrometer by infusion, in panel E on the QTRAP 2000 by LC/MS/MS, and in panel F on the MALDI-TOF-TOF mass spectrometer. The b and y ion masses in all panels are consistent with OP covalently bound to Tyr 238. (A) The doubly charged parent ion of the FP-biotin–labeled peptide is at 726.0 m/z. Masses enclosed in boxes at 227.3, 312.5, and 329.5 amu are fragments of FP-biotin. The immonium ion of FP-biotinylated tyrosine is at 708.8 amu. Its partner ion at 691.6 amu has lost 17 amu. (B) The doubly charged parent ion of the DFP-labeled peptide is at 522.0 m/z. Loss of one or both isopropyl groups during collision-induced dissociation yields y-ions minus 42 or minus 84 amu, confirming the presence of diisopropylphosphate. Loss of both isopropyl groups is the most common observation (Grigoryan et al., 2008; Li et al., 2007). The y-ion series (y1 and y2–84 through y6-84) indicates that tyrosine is labeled. The delta mass (242.9 amu) between y1 (147.2 amu) and y2–84 (390.1 amu) is consistent with the appearance of tyrosine phosphate at fragment y2 (163 amu for tyrosine and 80 amu for phosphate). The mass at 373.2 amu is the y2 ion minus 101 amu, representing loss of two isopropyl groups as well as ammonia. The mass at 501.8 m/z is the doubly charged parent ion minus one isopropyl group. The mass at 216.1 amu is consistent with the phosphotyrosine immonium ion. (C) The doubly charged parent ion of the chlorpyrifos oxon-labeled peptide is at 508.7 m/z. The y-ion series (y1–y6) shows the presence of all residues. The mass difference (299.0 amu) between y1 (147.2 amu) and y2 (446.2 amu) clearly shows the diethoxyphosphate on tyrosine in fragment y2 (163 amu for tyrosine and 136 amu for diethoxyphosphate). The mass at 244.1 amu is consistent with the monoethoxyphosphotyrosine immonium ion. The mass at 272.0 amu is consistent with the diethoxyphosphotyrosine immonium ion. (D) The doubly charged parent ion of the dichlorvos-labeled peptide is at 494.0 m/z. The y-ion series (y1–y6) shows the presence of all residues. The mass difference (270.9 amu) between y1 (147.2 amu) and y2 (418.1 amu) clearly shows the dimethoxyphosphate on tyrosine in fragment y2 (163 amu for tyrosine and 108 amu for dimethoxyphosphate). The mass at 244.0 amu is consistent with dimethoxyphosphotyrosine immonium ion. (E) The parent ion of the soman-labeled peptide has a mass to charge ratio of 520.7, but this mass does not appear in the scan. The prominent peak at 479.5 is the doubly charged parent ion that has lost the pinacolyl group from soman. The y2–y7 ions have all lost 84 amu due to release of pinacolyl from soman. The peak at 214.4 is the methylphosphotyrosine immonium ion. (F) The parent ion of the sarin-labeled peptide is the singly charged ion at 998.5 amu. The masses of the y-ion series, and the 214.1 mass for methylphosphotyrosine immonium, support labeling on tyrosine.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.
8.
FIG. 5.

FIG. 5. From: Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine.

MS/MS spectra of OP labeled Tyr 574 in peptide KPVEEYANCHLAR of human transferrin. Mass spectra in (A), (B), and (C) were acquired on the QTRAP 4000 mass spectrometer by infusion, in (D) on the MALDI-TOF-TOF, in panels E and F by LC/MS/MS on the QTRAP 2000 mass spectrometer. The b and y ion masses in all panels are consistent with OP covalently bound to Tyr 574. Cysteine has been CAM, which adds a mass of 57 amu. (A) The quadruply charged parent ion of the FP-biotin–labeled peptide is at 541.4 m/z. Masses enclosed in boxes at 227.3, 312.6, and 329.6 amu, are fragments of FP-biotin. The immonium ion of FP-biotinylated tyrosine is at 708.7 amu. Its partner ion at 691.6 amu has lost 17 amu. Three y-ions have lost 328 amu from FP-biotin. (B) The triply charged parent ion of the DFP-labeled peptide is at 584.2 m/z. Loss of one or both isopropyl groups yields ions minus 42 or minus 84 amu. The y-ion series (y1–y7) supports the identification of the peptide and indicates that the OP label is not in that portion of the peptide. The delta mass (243.2 amu) between y7 (841.3 amu) and y8-84 (1084.4 amu) fits with the appearance of tyrosine-phosphate in fragment y8 (163 amu for tyrosine and 80 amu for phosphate). The presence of tyrosine-phosphate is expected for tyrosine-diisopropylphosphate that has lost both isopropyl groups. Masses at 1213.7 (y9-84), 826.0 (b6-84) and 897.4 amu (b7-84) support this assignment. The mass at 215.9 amu is consistent with the phosphotyrosine immonium ion. (C) The triply charged parent ion of the chlorpyrifos oxon-labeled peptide is at 575.2 m/z. The y-ion series (y1–y11) shows a delta mass (299.4 amu) between y7 (841.3 amu) and y8 (1140.7 amu) that is consistent with the appearance of tyrosine-diethoxyphosphate at fragment y8 (163 amu for tyrosine and 136 amu for diethoxyphosphate). This is supported by the b-ion series (b1–b9) which shows the appearance of the same tyrosine as tyrosine-diethoxyphosphate at fragment b6. The mass at 272.5 amu is consistent with the diethoxyphosphotyrosine immonium ion. (D) The singly charged parent ion of the dichlorvos-labeled peptide is at 1694.9 m/z. This is a MALDI-TOF-TOF spectrum, where parent ions are typically singly charged. The y-ion series (y1–y10) shows a delta mass (271.1 amu) between y7 (841.6 amu) and y8 (1112.7 amu) which is consistent with the appearance of tyrosine-dimethoxyphosphate at fragment y8 (163 amu for tyrosine and 108 amu for dimethoxyphosphate). The mass at 244.1 amu is consistent with dimethoxyphosphotyrosine immonium ion. (E) The triply charged parent ion of the soman-labeled peptide has a mass of 583.9, but this mass does not appear in the spectrum. The mass at 555.5 is the triply charged parent ion that has lost 84 amu from the pinacolyl group of soman. The doubly charged y8–y13 ions have all lost 84 amu from soman; the y8–y13 masses support labeling on tyrosine. The methylphosphotyrosine immonium ion is present at 214.3 amu. (F) The quadruply charged parent ion for sarin-labeled peptide has a mass of 427.7, but this mass does not appear in the spectrum. The most prominent peak is the methylphosphotyrosine immonium ion at 214.3 amu, supporting labeling of tyrosine by sarin. The doubly charged y8 and y9 ions, and the triply charged b5 and b6 ions also support labeling on tyrosine. These ions have lost 42 amu due to loss of the isopropyl group from sarin.

Bin Li, et al. Toxicol Sci. 2009 January;107(1):144-155.

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