Characteristic fragments from OP labeled lysine
When chlorpyrifos-oxon reacts with lysine, the added mass is 136 amu (for diethoxyphosphate). Under CID conditions, there is a relatively facile gas-phase elimination of one or both ethylene side-chains (28 amu each) from the diethoxyphosphate [31
] resulting in a monoethoxyphosphate adduct (added mass 108 amu) or a phosphate adduct (added mass 80 amu). This side-chain elimination explains the lysine related masses observed during CID fragmentation of chlorpyrifos-oxon modified lysine-containing peptides (see ).
CID fragmentation of peptides containing diethoxyphosphate-modified lysine yields non-sequence, characteristic ions at 237, 220, 209, 192, 164, 130, 129, and 84 amu. These masses correspond to the diethoxyphosphate adduct of the lysine immonium ion (136 + 101 = 237 amu), the diethoxyphosphate adduct of the lysine immonium ion minus NH3 (136 + 84 =220 amu), the monoethoxyphosphate adduct of the lysine immonium ion (108 + 101 = 209 amu) the monoethoxyphosphate adduct of the lysine immonium ion minus NH3 (108 + 84 = 192), the phosphate adduct of the lysine immonium ion minus NH3 (80 + 84 = 164), pipecolic acid (130 amu), α-amino-caprolactam (129 amu), and the lysine immonium ion minus NH3 and minus all vestiges of the diethoxyphosphate label (84 amu). lists these fragments, shows their structures, indicates the frequency with which they appeared in the 21 MSMS spectra of chlorpyrifos-oxon modified peptides, and indicates their intensities relative to the most intense mass in each spectrum.
As indicated in , the 237 amu mass could be interpreted as either the diethoxyphospho-lysine immonium ion or the monoethoxyphospho α-amino-caprolactam. If the latter interpretation were correct, one might expect to see ions consistent with the diethoxyphospho α-amino-caprolactam (at 265 amu). No peak at 265 amu appeared in any of the 21 MSMS spectra, suggesting that the α-amino-caprolactam interpretation is incorrect. Conversely, masses for both the diethoxyphospho-lysine immonium ion (237 amu) and the monoethoxyphospho-lysine immonium ion (209 amu) were detected (see ), suggesting that the lysine immonium ion interpretation is correct. Modified immonium ions are most often the source of characteristic fragments [35
The most prevalent characteristic ion was the diethoxyphospho-lysine immonium ion minus (220 amu), appearing in 95% of the MSMS spectra (20 out of 21). This is consistent with NH3
to be a the results of Fenaille et al. who found the modified-lysine immonium ion minus NH3
prominent mass in MSMS spectra of hexanal-modified lysine containing peptides [33
]. Though the intensity of the 220 amu ion varied from 6% to 100%, of the most intense ion in the spectrum, its intensity was generally around 30%. On three occasions it was the most intense ion in the spectrum. The two next most common characteristic ions were also modified-derivatives of the lysine immonium ion minus NH3
: monoethoxyphospho-lysine immonium ion minus NH3
(163.9 amu). The lysine immonium (192.0 amu) and phospho-lysine immonium ion minus NH3
ion (without modification, 83.9 amu) and the diethoxyphospho-lysine immonium ion (237.1 amu) also were frequently present. Occasionally, masses for the α-amino caprolactam (128.9 amu), pipecolic acid (129.9 amu), and monoethoxyphospho-lysine immonium ion (209.1 amu) appeared.
Once identified, the characteristic ions for the diethoxyphosphate-adduct were particularly useful in that they provided a convenient way to identify diethoxyphosphate-labeled lysine in other peptides.
Only human serum albumin was treated with diisopropylfluorophosphate, and only one labeled peptide was found, LAK*TYETTLEK. Since no other proteins were treated with diisopropylfluorophosphate, it is entirely possible that other diisopropylfluorophosphate-labeled peptides may be found in the future.
When diisopropylfluorophosphate reacts with lysine, the added mass is 164 amu (for diisopropoxyphosphate). Under CID conditions, gas-phase elimination of the isopropylene side-chains (42 amu) is even more facile than elimination of ethylene is for the diethoxyphospho-adducts [31
]. Isopropylene elimination is so easy that there is no evidence for characteristic ions that retain a side chain.
CID fragmentation of the peptide containing the diisopropoxyphosphate-modified lysine yielded non-sequence, characteristic ions at 181, 164 and 84 amu. These masses correspond to the phospho-lysine immonium ion (80 + 101 = 181 amu), the phospho-lysine immonium ion minus NH3 (80 + 84 = 164 amu), and the lysine immonium ion minus NH3 and minus all vestiges of the diisopropoxyphosphate label (84 amu). lists these fragments, shows their structures, and indicates their intensities relative to the most intense mass in the spectrum. Since only one diisopropoxyphospho-peptide was identified, the frequency with which they appear in the MSMS spectra is set at 100%.
Illustration of CID fragmentation for peptides containing OP-lysine adducts
through show representative MSMS fragmentation spectra for peptides containing OP-labeled lysine. There is a spectrum for the diisopropoxyphosphate adduct (), a spectrum for a peptide carrying a single diethoxyphosphate adduct on lysine which yielded characteristic fragments (), a spectrum for a diethoxyphosphate adduct on lysine which did not yield characteristic fragments (), and a spectrum for a peptide carrying a diethoxyphosphate adduct on lysine and another diethoxyphosphate adduct on tyrosine which yielded fragments characteristic of both labeled amino acids ().
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 (more ...)
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. (more ...)
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] (more ...)
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 (more ...)
shows the MSMS spectrum of the diisopropoxyphosphate-labeled peptide LAK*TYETTLEK from human serum albumin. It is labeled on the lysine that is three residues from the N-terminus. That this lysine was not cleaved during the tryptic digestion supports the proposal that it is labeled. The parent ion is doubly-charged with an m/z of 731.0, which includes the mass of the amino acid sequence plus an added mass of 164 amu for the diisopropoxyphosphate.
The spectrum is complicated by the facile loss of isopropylene (42 amu) from the diisopropoxyphosphate [31
]. This neutral loss fragmentation accounts for the doubly-charged peaks at 709.8, and 688.3 amu, designated P1 and P2. These are associated with the parent ion (at 731.0 amu, designated P), and are consistent with the loss of first one and then both isopropylene groups from the diisopropoxyphosphate adduct of the parent ion.
A similar phenomenon occurs for the b3 ion. This ion appears in four forms, designated b3, b3a, b3b, and b3c. The mass of the b3c fragment (477.6 amu) is consistent with the N-terminal three amino acids, LAK, plus the mass of diisopropoxyphosphate. Fragment b3b is 42 amu smaller (at 435.6 amu) consistent with the loss of one isopropylene group. Fragment b3a (at 393.6 amu) is smaller by yet another 42 amu. Finally, the b3 fragment (at 313.4 amu) is 80 amu smaller than b3a indicative of the loss of the phospho-moiety. The masses of these b3 fragments are all consistent with diisopropoxyphosphate labeling of the lysine at position 3.
The b-series, without any added mass, continues from b3 to b5. In addition, there is a y-series that extends from y2 to y8. None of the residues in this series show any indication of being labeled. The y-series includes residues y7 and y8, which carry the two other potentially reactive groups, threonine and tyrosine. The fact that their masses do not include the adduct mass indicates that they are not labeled. Most of the other major peaks in the spectrum are consistent with loss of water from the sequence ions or with characteristic fragments. The foregoing observations: the parent ion mass, the neutral losses from the parent ion, the sequence data, and the neutral loss from the b3 fragment clearly identify this peptide as being labeled by diisopropoxyphosphate on lysine.
Having established the identity of the label, we can turn to the non-sequence fragments in the CID spectrum to identify masses that are characteristic of this particular label. Three such masses appear in . They are enclosed in boxes for emphasis. The most intense of these masses is at 164.0 amu. It is consistent with phospho-lysine immonium ion minus NH3. The phospho-lysine immonium ion is also present (at 181.2 amu), as is the lysine immonium ion at 83.6 amu. Structures for these compounds are given in .
shows the MSMS spectrum of the diethoxyphosphate-labeled peptide STTK*DLLFR from mouse transferrin. It is labeled on the lysine that is four residues from the N-terminus. That this lysine was not cleaved during the tryptic digestion supports the proposal that it is labeled. The parent ion is doubly-charged with an m/z of 609.0, which includes the mass of the amino acid sequence plus an added mass of 136 amu for the diethoxyphosphate.
A y-ion series from y1 to y7 is present. The delta mass for the diethoxyphosphate-lysine adduct appears in the sequence between y5 and y6. This delta mass is 263.9 amu, consistent with the expected value for lysine (128 amu) plus diethoxyphosphate (136 amu). Unlike the diisopropoxyphosphate adduct, neutral loss is not evident from either the parent ion or the sequence ions. This is similar to our experience with modified tyrosine [31
]. The masses at 464.8 amu and 514.8 amu are consistent with doubly-charged forms of y6 and y7. Most other significant masses could be assigned as fragments due to loss of ammonia from sequence ions, to loss of water from the parent ion, to internal fragments or characteristic ions. The parent ion mass and the sequence data, including the y5–y6 interval for diethoxyphospho-lysine, clearly identify this peptide as being labeled on lysine by diethoxyphosphate.
Non-sequence, characteristic fragments appeared at 237.5 amu (diethoxyphospho-lysine immonium ion), 220.4 amu (diethoxyphospho-lysine immonium ion minus NH3
), 209.4 amu (monoethoxyphospho-lysine immonium ion), 192.3 amu (monoethoxyphospho-lysine immonium ion minus NH3
) and 164.2 amu (phospho-lysine immonium ion minus NH3
). Though neutral loss of ethylene (28 amu) from neither the parent ion nor the sequence ions was observed, neutral loss from the immonium ion seems to be more facile. A similar observation was made for diethoxyphospho-tyrosine [31
]. Structures of the characteristic ions are given in .
shows the MSMS spectrum of the diethoxyphosphate-labeled peptide VAPEEHPTLLTEAPLNPK*ANR from bovine actin. It is labeled on the lysine that is four residues from the C-terminus. That this lysine was not cleaved during the tryptic digestion supports the proposal that it is labeled. The parent ion is triply-charged with an m/z of 811.9, which includes the mass of the amino acid sequence plus an added mass of 136 amu for the diethoxyphosphate.
A y-ion series from y2 to y11 is present. There is a gap in the sequence at y4, which is the position of the proposed lysine adduct. The delta mass for this gap is too large for simply the lysine adduct. However, it is consistent with the interval between y3 and y5 (361.3 amu) which would include lysine plus diethoxyphosphate plus proline (128 + 136 + 97 = 361 amu). This places the diethoxyphospho-adduct on either lysine or proline. Since proline is not a viable candidate for labeling, it may be concluded that the label resides on lysine. In addition to the y-series, there are strong peaks for the b12 and b13 fragments, both the singly-charged forms (at 1317.5 and 1388.5 amu) and the doubly-charged forms (at 659.2 and 694.8 m/z). Most other major peaks can be assigned to internal fragments.
The parent mass and the sequence data, including the y3–y5 interval for diethoxyphospho-lysine and proline, clearly identify this peptide as being labeled by diethoxyphosphate on lysine. However, there is no evidence for characteristic ions in the MSMS spectrum. This was the only peptide of the 21 CPO-labeled peptides we analyzed that did not show any characteristic fragments.
shows the MSMS spectrum of the diethoxyphosphate-labeled peptide K*Y*VPR from bovine tubulin beta. This peptide is doubly-labeled, once on the N-terminal lysine and again on the neighboring tyrosine. In support of the foregoing statement, the parent ion mass is 467.5 m/z (doubly-charged), which includes the mass of the amino acid sequence plus two-times the added mass of 136 amu, for the two diethoxyphosphate labels. The N-terminal lysine and the neighboring tyrosine are the only reasonable candidates for labeling in this peptide. Simultaneous labeling of both lysine and tyrosine occurred in four other peptides (): Y*AK*R, LSVDY*GK*K and FDLMY*AK*R (all three from bovine tubulin alpha) and Y*TKK*VPQVSTPTLVEVSR (from human serum albumin). Thus, double-labeling is not an isolated phenomenon. Labeling of two tyrosines in a single peptide was observed for two peptides from mouse transferrin [31
A complete y-ion series for K*Y*VPR is present in . The interval between y3 and y4 (299.3 amu), is consistent with a diethoxyphosphate-labeled tyrosine (163 + 136 = 299 amu). Addition of 264 amu to y4 (670.3 amu) yields the singly-charged parent ion mass of 934 amu. The mass for diethoxyphosphate-labeled lysine is 264 amu (128 + 136 amu). There is a neutral loss from y4 of 28 amu (to give the peak at 642.2 amu) which is most likely due to dissociation of ethylene from the diethoxyphosphate adduct on tyrosine. A similar loss of 28 amu from b3 (to give the peak at 635.0 amu) is more difficult to interpret. B-ions readily lose CO (28 amu) to yield a-ions. Alternatively, the 28 amu loss could again reflect loss of ethylene. The 635.0 amu mass has been designated a3 in .
Characteristic ions for diethoxyphospho-lysine appear at 220.0, 191.9, 163.9, and 83.9 amu. In addition, there is a mass at 236.9 amu which could be interpreted as the diethoxyphospho-lysine immonium ion or the a1 ion. There is a significant peak at 244.0 amu, which is consistent with the monoethoxyphospho-tyrosine immonium ion [31
]. A more commonly observed characteristic ion for chlorpyrifos oxon labeled tyrosine is the diethoxyphospho-tyrosine immonium ion at 272 amu [31
]. Though an intense peak exists at 271.9 amu, its interpretation is complicated by the possibility of a proline-arginine internal fragment (nominal mass of 272 amu). Support for the existence of the internal fragment is the 97.7 amu mass which is consistent with the N-terminal proline expected from such an internal fragment. At a minimum, the 271.9 amu mass is a combination of the proline-arginine internal fragment and the diethoxyphospho-tyrosine immonium characteristic ion.
A final complication in this MSMS spectrum is a group of doubly-charged fragments (at 453.5, 414.0, 399.6 and 391.1 m/z) in the vicinity of the parent ion. The 453.5 m/z fragment is 28 amu smaller than the parent ion, making it tempting to assign 453.5 to a neutral loss of ethylene from the parent ion. Similarly, the 399.6 m/z fragment is 136 amu smaller than the parent, making it reasonable to assign 399.6 to a neutral loss of the entire diethoxyphosphate group, i.e. HPO(OCH2CH2)2. However, a neutral loss of this sort has not been seen before, either for diethoxyphospho-tyrosine or diethoxyphospho-lysine adducts. The m/z 414.0 is still more confusing. Being 108 amu smaller than the parent, it would appear to be due to neutral loss of monoethoxyphosphate where the second ethylene remains attached to the peptide; however, we can conceive of no mechanism to accommodate such an interpretation. The 391.1 m/z is a simple loss of 17 amu (NH3) from 399.6 m/z. One might attribute these masses to contaminants or some other sort of artifact except that an analogous pattern of peaks is seen in the MSMS spectrum of Y*AK*R (data not shown).
The parent ion mass, the sequence data (including the y3 to y4 and y4 to parent ion intervals), and the characteristic fragments strongly argue that this peptide is doubly-labeled by chlorpyrifos-oxon, carrying diethoxyphosphate groups on both lysine and tyrosine.