FP-Biotin Labeled Albumin in Human Plasma
The structures of the organophosphorus agents are shown in Figure . Five tyrosines and two serines in human albumin were labeled with FP-biotin including Tyr 138, Tyr 148, Tyr 401, Tyr 411, Tyr 452, Ser 232, and Ser 287 (Table ).
Figure 1 Structures of organophosphorus agents. Covalent binding to tyrosine or serine results in loss of the fluoride ion from soman, DFP, and FP-biotin and of the aromatic ring from CPO, so that the added mass is 162.2 for soman, 164.1 for DFP, 136.0 for CPO, (more ...)
FP-Biotinylated Human Albumin Tryptic Peptides Identified by LC/MS/MSa
Supporting MS/MS spectra for these assignments are in Figures −. A peptide labeled with FP-biotin had ions at 227, 312, and 329 atomic mass units (amu) resulting from fragmentation of FP-biotin (12
). Two ions characteristic of covalent binding of FP-biotin to the hydroxyl group of tyrosine are the immonium ion of tyrosine-FP-biotin at 708 amu and its partner ion at 691 amu, produced by loss of NH3
. The 708 and 691 amu masses are prominent in Figure A,B but barely visible in Figure A,B. An additional complexity in Figure A is the presence of ions that had lost a 329 or 226 amu fragment from FP-biotin.
Figure 2 MS/MS spectra of albumin peptides labeled with FP-biotin on Tyrosine. (A) Tyr 411 in peptide YTK and (B) Tyr 138 in peptide YLYEIAR are covalently modified by FP-biotin. The characteristic fragments of FP-biotin at 227.2, 312.4, and 329.3 amu are present. (more ...)
Figure 6 MS/MS spectrum of albumin peptide labeled with FP-biotin on Ser 232. This spectrum was acquired on the QSTAR elite mass spectrometer. The doubly charged parent ion is at 726.9 amu. The peak at 591.3 is FP-biotin released from serine, carrying a hydroxyl (more ...)
Figure 3 MS/MS spectra of albumin peptides labeled with FP-biotin on Tyrosine. (A) Tyr 148 in peptide HPYFYAPELLFFAK is labeled with FP-biotin. The quadruply charged parent ion has a mass of 579.7 m/z. The FP-biotin tyrosine immonium ion is at 708.5; after neutral (more ...)
The masses in Figure A are consistent with the sequence YTK where the added mass of 572 amu from FP-biotin is on Tyr. The complete y-ion series is present (y1, 147.0 amu, Lys; y2, 248.4 amu, LysThr; and the doubly charged, FP-biotinylated parent ion). Peaks at 227.2, 312.4, 329.4, 691.3, and 708.5 amu are indicative of the presence of FP-biotinylated tyrosine. The remaining major peaks are consistent with various FP-biotinylated tyrosine fragments missing pieces of the FP-biotin moiety.
Peptide YLYEIAR has two tyrosines. A y-ion series (y1−y6) indicates that the FP-biotin label is on the N-terminal Tyr. Additional evidence for labeling on Tyr 138 rather than on Tyr 140 was the presence of the a2 ion at 821.4 amu, the b2 ion at 849.2 amu, the a1+2 ion at 354.8, and the a2+2 ion at 411.4 m/z (Figure B). If the FP-biotin had been attached to Tyr 140, the masses would have been a2 = 249, b2 = 277, a1+2 = 68, and a2+2 = 125 amu. Peaks at 226.3, 312.4, and 329.3 are fragments of FP-biotin. Masses at 708.2 and 691.3 amu for FP-biotinylated tyrosine confirm the presence of FP-biotinylated tyrosine in the peptide. Analysis of the missed cleavage peptide KYLYEIAR supports labeling on Tyr 138 (data not shown).
Peptide HPYFYAPELLFFAK in Figure A also has two tyrosines. Evidence for labeling on Tyr 148 rather than on Tyr 150 is the presence of the b4 ion at 1118.5 amu, the a4+2 ion at 545.4 amu, and the b4+2 ion at 559.3 m/z. The total mass of the b4 fragment (1117.6 amu) is equal to the fragment HPYF (545 amu) plus the added mass from FP-biotin (572 amu). Of the four residues in the b4 fragment, Tyr 148 is the most reasonable candidate for labeling. Fragment masses for b5 and b6 also support labeling of Tyr 148 rather than Tyr 150. An extensive y-ion series (y2−y8) supports the assignment of this peptide. Masses at 227.3, 312.2, and 329.4 indicate the presence of FP-biotin. Masses at 691.2 and 708.5 amu indicate the presence of FP-biotinylated tyrosine. A similar analysis was made for peptides RHPYFYAPELLFFAK and HPYFYAPELLFFAKR, which differ from HPYFYAPELLFFAK by virtue of missed cleavages (data not shown).
Peptide QNCELFEQLGEYK in Figure B is FP-biotinylated on Tyr 401 as demonstrated by the y2 ion at 882.5 amu, the y3 ion at 1011.5 amu, the y4 ion at 1068.5 amu, and the y5 ion at 1181.8 amu. The y2 mass is equal to the sum of Lys (147 amu), Tyr (163 amu), and the added mass of FP-biotin (572 amu). A variety of larger, multiply charged y-ion fragments support the labeling assignment. Prominent b-ion fragments confirm the identity of the peptide. Fragments at 227.2, 312.2, and 329.3 amu indicate the presence of FP-biotin in the peptide.
Peptide MPCAEDYLSVVLNQLCVLHEK in Figure is FP-biotinylated on Tyr 452. The best evidence in support of this interpretation is the doubly charged mass at 720.4 m/z, which is consistent with the b7 ion plus the added mass from FP-biotin. The b7 ion consists of MPCAEDY. Of these residues, only Tyr 452 is a reasonable candidate for FP-biotinylation. The b8+2, b9+2, and b10+2 ions support this interpretation. The y-series (y3−y11) supports identification of this peptide. Masses at 227.2, 312.2, and 329.4 indicate the presence of FP-biotin. Masses at 691.4 and 708.4 amu indicate the presence of FP-biotinylated tyrosine in this peptide. A missed cleavage form of this peptide, RMPCAEDYLSVVLNQLCVLHEK, was also analyzed, and the results support labeling of Tyr 452 (data not shown).
Figure 4 MS/MS spectrum of albumin peptide labeled with FP-biotin on tyrosine 452. The quadruply charged parent ion has a mass of 773.3 m/z. The carbamidomethylated cysteine is indicated as CAM. Internal fragmentation at proline yielded the 458.2 ion for PC(CAM)AE (more ...)
Peptide SHCIAEVENDEMPADLPSLAADFVESK in Figure A is FP-biotinylated on Ser 287. Existence of an FP-biotinylated serine is indicated by the major peak at 591.4 amu. This is a characteristic mass from FP-biotin that appears as the result of β-type elimination of FP-biotin from a serine adduct (Figure B), during collision-induced dissociation in the mass spectrometer (12
). The complementary peptide fragment arising from this fragmentation contains a dehydroalanine in place of serine. The masses of a b-series (Δb5−Δb12) containing a dehydroalanine residue support the elimination of FP-biotin from serine. Of the residues in the b5 fragment (SHCIA), serine at position 287 is a candidate for FP-biotinylation. The cysteine might have been considered a target for labeling, but the overall mass of the fragment is consistent with carbamidomethylation on the cysteine. A y-ion series (y4−y15) supports the identification of the peptide. Additional support for the presence of FP-biotin in the peptide comes from characteristic masses at 312.1 and 329.2 amu. The absence of the characteristic mass at 227 amu is common for FP-biotinylated serine.
Figure 5 (A) MS/MS spectrum of albumin peptide labeled with FP-biotin on Ser 287. The triply charged parent ion has a mass of 1183.8 m/z. The carbamidomethylated (CAM) peptide carried the FP-biotin on Ser 287. The evidence for modification on serine is the presence (more ...)
The MS/MS spectrum for peptide AEFAEVSK labeled by FP-biotin on Ser 232 is in Figure . The b- and y-ion masses support the assigned sequence. Peaks not assigned by Protein Pilot included six dehydroalanine fragments as well as the 591 amu ion of FP-biotin and the 227, 312, and 329 amu fragments of FP-biotin. These additional peaks strongly support the conclusion that Ser 232 of albumin was labeled by FP-biotin. This labeled peptide was detected by the sensitive QSTAR elite mass spectrometer but not by the QTRAP 2000 mass spectrometer. No FP-biotinylated peptides were found in the control plasma that had not been treated with FP-biotin.
Search for Other FP-Biotin-Labeled Proteins in Human Plasma
The present method identified 7 FP-biotin-labeled albumin peptides but no FP-biotin-labeled peptides from any other protein. A Western blot hybridized with Streptavidin Alexafluor-680 showed many FP-biotinylated bands in human plasma treated with FP-biotin under our conditions (data not shown). One such protein is FP-biotinylated plasma butyrylcholinesterase (1
). However, the FP-biotinylated butyrylcholinesterase peptide was not found with the present methods. FP-biotinylated peptides from proteins other than albumin are difficult to detect in the presence of the overwhelmingly high concentration of albumin. Even after depletion of albumin with Cibacron Blue, the concentration of albumin was still too high to allow detection of other FP-biotinylated peptides. Human plasma contains 5 mg of butyrylcholinesterase and 50000 mg albumin per L. In experiments not described in this report, we found OP-labeled butyrylcholinesterase in human plasma only after the butyrylcholinesterase had been purified by binding to procainamide affinity gel, thus eliminating more than 95% of the albumin.
Albumin Residues Labeled by CPO
Prolonged labeling of pure human albumin with CPO resulted in labeling of six tyrosines: Y138, Y150, Y161, Y401, Y411, and Y452 (Table ). Four of these sites were also labeled by FP-biotin (Y138, Y401, Y411, and Y452). The HPYFYAPELLFFAK peptide was labeled on Tyr 150 by CPO, whereas it was labeled on Tyr 148 by FP-biotin. A new peptide YKAAFTECCQAADK was labeled by CPO (Figure ) and not by FP-biotin. Labeling on tyrosine is supported by the b ion series. The identity of the peptide is supported by the y2−y8 ions. Additional MS/MS spectra for CPO-labeled peptides are in the Supporting Information
CPO-Labeled Human Albumin Peptidesa
MS/MS spectrum of albumin peptide labeled with CPO on Tyr 161. The b2 and b3 ions support labeling on tyrosine.
Tyr 411 Reacts Most Readily with OP
The finding that seven tyrosines and two serines make a covalent bond with OP led to the question of which amino acid reacts most readily with OP. To answer this question, we duplicated the conditions reported to label one molar equivalent of human albumin with DFP (10
). MALDI-TOF analysis of pepsin-digested, DFP-treated human albumin suggested that 80% of Tyr 411 was labeled with DFP. MS/MS analysis of a tryptic digest of carbamidomethylated DFP-treated albumin confirmed that Tyr 411 in peptide Y*TK was labeled. In addition, less than 10% of peptide EFNAETFTFHADICT*LS*EK was labeled (on residues T515 and S517).
Albumin treated with FP-biotin for 2 h and digested with pepsin had 52% of its Tyr 411 labeled in peptide VRY*TKKVPQVSTPTL as calculated by MALDI-TOF mass spectrometry (Figure ). The carbamidomethylated, trypsin-digested preparation analyzed by LC/MS/MS confirmed that Tyr 411 in peptide Y*TK was labeled with FP-biotin. Peptide HPY*FYAPELLFFAK was labeled on Tyr 148 with FP-biotin but to less than 10%. A third method to identify FP-biotinylated peptides was purification on monomeric avidin beads followed by MALDI-TOF-TOF analysis. This method yielded only one FP-biotinylated peptide, the Y*TK peptide labeled on Tyr 411.
Figure 8 MALDI-TOF spectrum of pepsin-digested albumin to show labeling of Tyr 411 by FP-biotin. Pepsin digestion of albumin yields two unlabeled peptides at 1717.2 (VRYTKKVPQVSTPTL) and 1830.3 (LVRYTKKVPQVSTPTL) amu, both containing Tyr 411. Both peptides have (more ...)
Soman-treated albumin (150 μM soman for 2 h) analyzed by MALDI-TOF and LC/MS/MS yielded only one labeled peptide. The soman was on Tyr 411.
Albumin treated with CPO for 2 h before digestion with pepsin or trypsin and analyzed by MALDI-TOF and LC/MS/MS was labeled on Tyr 411. Approximately 30% of the Tyr 411 sites were labeled in peptides VRY*TKKVPQVSTPTL and LVRY*TKKVPQVSTPTL. In addition, less than 5% of Thr 566 in peptide ET*CFAEEGKK and less than 5% of Thr 236 and Thr 239 in peptide LVT*DLT*KVHTECCHGDLLECADDR were labeled. We conclude that Tyr 411 is the most OP reactive residue in human albumin.
Support for the conclusion that Tyr 411 is the most OP reactive residue in albumin comes from ref (2
). Williams incubated the albumin fraction of human plasma with radiolabeled sarin, digested with trypsin, purified the radiolabeled peptides by HPLC, and analyzed by LC/MS/MS. A single radiolabeled peptide was isolated. Its sequence was YTK with the isopropyl methylphosphonyl group on tyrosine.
Unstable OP-Ser and OP-Thr but Stable OP-Tyr
It was noted that serine and threonine residues were labeled in addition to tyrosine when samples had been incubated at pH 8.0−8.3 for 2−48 h but were not found in samples incubated at pH 8.3 for a month. In contrast, OP-labeled tyrosines were found even after 1 month of incubation at pH 8.3. Our stability study of CPO-labeled albumin confirmed that the Tyr 411 adduct was stable (Figure ). Incubation at pH 7.4 and 22 °C resulted in almost no loss of the CPO label on Tyr 411 after 7 months. In contrast, about half of the label was lost after 3.6 months at pH 8.3 and 22 °C. The CPO-labeled Tyr 411 was stable at pH 1.5 and 22 °C and was stable at all pH values when the labeled albumin was stored at −80 °C. These results suggest that OP-labeled serine and threonine adducts are unstable as compared to OP-labeled tyrosine.
Figure 9 Stability of the diethoxyphosphate adduct of human albumin on Tyr 411. Albumin was treated with CPO to achieve 97% labeling of Tyr 411. Excess CPO was removed by dialysis. The pH of the dialyzed albumin was adjusted to 1.5, 7.4, and 8.3. CPO-albumin samples (more ...)
Surface Location of OP Reactive Residues
The crystal structure in Figure shows the five tyrosines and two serines that become labeled by FP-biotin. These residues are located on the surface of the albumin molecule where they are available for reaction with OP.
Figure 10 Crystal structure of human albumin showing surface location of Tyr 138, Tyr 148, Tyr 401, Tyr 411, Tyr 452, Ser 232, and Ser 287. The residues are shown as space-filled structures. The picture was made with PyMol software using the structure in PDB code (more ...)
Human albumin has 18 tyrosines and 24 serines but only five tyrosines and two serines made a covalent bond with FP-biotin. Their special reactivity may be explained by a nearby arginine or lysine that stabilizes the ionized hydroxyl of tyrosine or serine.