3.1. Strategy for identifying labeled residues in tubulin
The strategy is to first use FP-biotin to label the tubulin. FP-biotinylated peptides are easy to find because the biotin tag gives a signature fragmentation pattern. Masses of 227, 312, and 329 amu are always present in the MS/MS scan of an FP-biotin labeled peptide [9
]. We use the MS/MS function of the MALDI-TOF-TOF mass spectrometer to screen for FP-biotin labeled peptides. Then we use the Q-Trap 4000 mass spectrometer to fragment the peptides for de novo sequencing to identify the site of covalent attachment of FP-biotin.
In a second phase, the protein is labeled with other OP. In the first round of screening for peptides labeled with these other OP, the assumption is made that the sites labeled by FP-biotin are also labeled by other OP. This assumption allows one to calculate theoretical OP-peptide masses and to look for the presence of these masses in the HPLC-fractionated, tryptic digest using the MALDI-TOF-TOF mass spectrometer. However, this assumption may not hold for all OP. Therefore a second strategy is used. Peptide masses observed in the MS scan for OP-labeled peptides are compared with theoretical masses for unlabeled peptides. The list of theoretical masses is generated with Protein Prospector software (UCSF). This free software is available at http://prospector.ucsf.edu
. Candidates for OP-labeled peptides are chosen when their masses are equal to the sum of the known peptide mass and the added mass from the OP. These putative, OP-labeled peptides are further tested by CID fragmentation in the Q-Trap 4000 mass spectrometer, followed by manual de novo sequencing to identify the site of covalent, OP attachment.
3.2. Four tubulin peptides are labeled by OP
The structures of the OP studied in the present report are shown in . A portion of each OP and the phenolic proton from the labeled tyrosine are displaced when the OP makes a covalent bond with tubulin, so that the mass added to tubulin is less than the mass of the OP. The added masses are 120 amu for sarin, 136 amu for CPO, 162 amu for soman, 164 amu for DFP, and 572 amu for FP-biotin. The leaving group is fluoride ion for sarin, soman, DFP and FP-biotin, and is 3,5,6-trichloro-2-(O)-pyridine for CPO.
shows the tubulin tryptic peptides that are targets for OP-labeling, before (panel A) and after treatment with OP (panels B–F). The peaks at 534.4, 597.4, 738.5 and 808.5 m/z in panel A are unlabeled peptides with the sequences YVPR, TGTYR, GSQQYR, and EEYPDR. After treatment of tubulin with OP, new peaks appear whose masses correspond to some of the expected, theoretical masses for OP-labeled peptides ().
Figure 2 Mass spectra of tryptic peptides of bovine tubulin, before and after labeling with OP. Peptides from A) control bovine tubulin, B) sarin treated tubulin, C) CPO treated tubulin, E) DFP treated tubulin, D) soman treated tubulin, F) FP-biotin treated tubulin. (more ...)
Theoretical masses of bovine tubulin tryptic peptides covalently labeled by OP
Unlabeled active site peptides were present in each digest, indicating that modification by OP was incomplete. The relative amount of labeled and unlabeled peptide was calculated from isotope cluster areas. The results are summarized in . FP-biotin, DFP, and CPO reacted with all 4 peptides, whereas sarin and soman reacted with only one peptide. Soman and sarin concentrations were significantly lower than the concentrations of the other OP during the labeling reaction, which might explain why fewer peptides were labeled.
Percent of each peptide labeled by OP
3.3. Tyrosine covalently modified by OP
Collision induced fragmentation in the Q-Trap mass spectrometer conclusively identified the amino acid sequence of each labeled peptide and the residue covalently modified by OP. The MS/MS spectra in , , , and show the y ions of each unlabeled peptide (panel A), and the same peptide after covalent modification by sarin, CPO, soman, DFP, and FP-biotin (panels B–F). The masses exactly fit the indicated sequence and fit the interpretation that the OP is attached to tyrosine ().
Figure 3 MS/MS spectra of the TGTYR peptide of alpha tubulin. A) Singly charged y ions derived from the doubly-charged, unlabeled parent ion at 299.3 m/z are shown. B) CPO-labeled TGTYR has a doubly charged parent ion of 367.4 m/z. The y ion masses are consistent (more ...)
Figure 4 MS/MS spectra of the YVPR peptide of beta tubulin. A) Singly charged y ions derived from the doubly charged, unlabeled parent ion at 267.2 m/z are shown. B) CPO-labeled YVPR has a doubly charged parent ion of 335.4 m/z. The y ion masses are consistent (more ...)
Figure 5 MS/MS spectra of the GSQQYR peptide of beta tubulin. A) Singly charged y ions derived from the doubly charged, unlabeled parent ion at 370.4 m/z are shown. B) Sarin-GSQQYR had a doubly charged parent ion at 429.4 m/z and the immonium methylphosphotyrosine (more ...)
Figure 6 MS/MS spectra of the EEYPDR peptide of beta tubulin. A) Singly charged y ions derived from the doubly charged, unlabeled parent at 404.3 m/z are shown. B) CPO-EEYPDR has a doubly charged parent ion at 472.3 m/z. The immonium phosphotyrosine ion is at (more ...)
OP-labeled tyrosines in bovine tubulin
Ions at 214, 216, 244, and 272 m/z provide additional evidence that the OP bind to tyrosine (see the figure legends for details).
3.4. Fragmentation patterns characteristic of a particular OP
The data from , , and reveal CID fragmentation patterns that are characteristic of particular OP. For example, DFP-peptides readily release one or both isopropyl to yield y ions missing either 42 or 84 amu. CPO-labeled peptides yield intense peaks at 216, 244 and 272 m/z that are consistent with phosphotyrosine immonium ion, phosphotyrosine and monoethylphosphotyrosine, respectively. The FP-biotinylated peptides release the characteristic fragments of FP-biotin at 227, 312, and 329 m/z. Singly charged ions missing either 227 or 329 amu were common. The pinacolyl group of soman was released from soman labeled YVPR peptide () to yield ions missing 84 amu. The isopropyl group of sarin was released from sarin labeled GSQQYR peptide () to yield ions missing 42 amu. Peptides that had lost 42 amu fragmented further to release NH3 (17 amu), yielding an ion pair characteristic of sarin labeling.
In no case was the entire OP released from tyrosine. The phosphate group remained bound to tyrosine during CID fragmentation. This contrasts with OP bound to serine where the fragmentation process releases the entire bound OP, leaving no trace of the OP behind, and yielding dehydroAlanine in place of the OP-labeled serine [12
3.5. No aging
When soman, sarin, or DFP are bound to acetylcholinesterase or butyrylcholinesterase they rapidly lose an alkyl group in a process called aging [13
]. An aged soman labeled peptide would have an added mass of 78 amu rather than 162; an aged sarin labeled peptide would have an added mass of 78 amu rather than 120; an aged DFP labeled peptide would have an added mass of 122 amu rather than 164 in the MS spectrum. No evidence of aging was found in OP labeled tubulin peptides as no masses representing aged OP-peptides were found in MS scans. We conclude that tubulin OP adducts on tyrosine do not age.