MS/MS spectra can provide different levels of information concerning
the methylation of transducer peptides
Fig. 3.27 shows MS/MS spectra, which were attributed via Mascot to different forms of the Htr4 peptide DEMSATIEEVAASA. The MS/MS spectra were recorded within the time of elution of the different molecular species shown in Fig. 3.25A and B. This provided the basis for the attribution of the MS/MS-identified peptide forms to these integrated MS spectra. Whereas spectra A and B of Fig. 3.27 show most of the peaks well above the background level, spectra C
Table 3.4. Mascot search result for the MS/MS fragmentation of the singly methylated Htr14-peptide DTVSATVEEIAASAN which was specified in the peptide list of Table 3.3.
Monoisotopic mass of neutral peptide Mr(calc): 1490.71 Variable modifications: E8 Methyl ester (DE)
Ions Score: 70 Expect: 5.6e-008
Matches (Bold Red): 83/215 fragment ions using 107 most intense peaks
# a a0 b b0 Seq. y y0 # 1 88.04 70.03 116.03 98.02 D 15 2 189.09 171.08 217.08 199.07 T 1376.69 1358.68 14 3 288.16 270.14 316.15 298.14 V 1275.64 1257.63 13 4 375.19 357.18 403.18 385.17 S (1176.57) 1158.56 12 5 446.22 428.21 474.22 456.21 A 1089.54 1071.53 11 6 547.27 529.26 575.27 557.26 T (1018.51) 1000.49 10 7 646.34 628.33 674.34 656.32 V 917.46 899.45 9 8 789.40 771.39 [b8': 803.39]817.39 [b80': 785.38]799.38 Em 818.39 800.38 8 9 918.44 900.43 946.44 928.43 E [y7': 689.33]675.33 [y70': 671.32]657.32 7 10 1031.53 1013.51 1059.52 1041.51 I 546.29 528.28 6 11 1102.56 1084.55 1130.56 1112.55 A 433.20 415.19 5 12 1173.60 1155.59 1201.59 1183.58 A 362.17 344.16 4 13 1260.63 (1242.62) 1288.63 1270.62 S 291.13 273.12 3 14 1331.67 1313.66 1359.66 1341.65 A 204.10 2 15 N 133.06 1
The list contains the calculated fragment masses for this peptide in case of a methylation at E8. Matches to the experimentally determined fragment masses are shown in red. A manual evaluation of the MS/MS spectrum showed that the values in parentheses were incorrectly assigned by the used algorithm. The b'- and y'-fragment masses which would be different for a methylation at E9 instead of E8 were manually added in brackets. The superscript zero indicates the loss of a water molecule (18 Da) from the corresponding fragment. The corresponding MS/MS spectrum is shown in Fig. 3.28D.
and D are of poorer quality and display a high level of background signals. However, as the elution times of the corresponding species are in agreement with the finding that per added methyl group the peptides elute approx. 2 - 3 min later, even the spectra of lower quality were considered to have reliably identified the peptide species.
Unambiguous identifications of the methylation positions in peptides can be based on either (i) the presence of significant mass peaks only for a methylation at one and not for the alternative methylation at the other residue of a methylation site or (ii) the complete absence of another methylatable residue within the complete peptide or at the identified methylation site.
The case of two potentially methylatable residues at a methylation site is shown in Fig. 3.27B. Here the presence of significantly higher peaks corresponding to the masses of fragments y6 and b8, as compared to peaks corresponding to the masses of fragments y6' and b8' (which would be expected in case of a methylation at the other Glu of the pair), argues for a methylation exclusively at or at least with a strong preference for the indicated position. Similar situations are depicted for two peptides from Htr14 (Fig. 3.28C and D). In both cases the MS/MS spectra unambiguously identified the exclusive (or strongly preferred) methylation position in the respective peptide.
Examples of unambiguous identifications of the methylation position due to the absence of a second methylatable residue are shown in Fig. 3.28A and B. The detection of the methylated form of Htr4 peptide DAAAEQATTLS is of particular interest, because it demonstrates that this peptide includes a physiological methylation site, which contains an Ala residue (at heptad position 2), a finding that was unprecedented. This might shed new light on the fact that Perazzona and Spudich (1999) saw a methylation in Htr1 only for the mutated methylation site containing the residues Glu265Ala266 but not for Ala265Glu266. At the first glance, the fact that methylation is seen in Htr4 at the site Ala738Glu739 seems to contradict the finding that in Htr1 methylation was only seen for the reverse order of residues. However, the investigated sites are located within the N-terminal methylatable region of Htr1, and the C-terminal methylatable region of Htr4, respectively. The methylatable Glu is therefore in both cases pointing away from the signaling region, so that the Ala-containing methylatable sites have the same spatial orientation within the transducer dimers. The detection of a methylation in Htr2 peptide DEVAGISQETAAQATAVA (obtained from the ∆cheB mutant WFS101) points to a second methylation site in addition to the one identified by Perazzona and Spudich (1999), and might explain why they saw some remaining methylation after mutating the methylation site Glu513Glu514 to a pair of alanines.
Figure 3.27 MS/MS spectra of the differently methylated forms of peptide DEMSATIEEVAASA of Htr4.
Each spectrum was produced by fragmentation of the respective peptide species and was recorded within the indicated interval of retention times (rt). All signals which correspond to the masses of predicted a-, b- or y- fragments are annotated. Arrows above and below the peptide sequence indicate the presence of signals for the indicated y- and b-fragments. A subscript m marks a methylated residue. Intensities are given relative to the base peak (here b2 which is cut off at 50%). Primed fragment designations in combination with dotted arrows indicate the
height of signals present at positions that would be calculated if the other glutamate were the methylated residue within the pair. Spectra are shown for the unmethylated (A), singly methylated (B) and doubly methylated (C) peptide species identified in strain S9 and for the doubly methylated species from ∆cheB strain WFS101 (D).
A
D B
Figure 3.28 Unambiguous MS/MS identifications of the methylated glutamate residue in different peptides.
Spectra were recorded and annotated as described in Fig. 3.27. Identifications of the methylated positions are unambiguous, either due to the absence of a second glutamate at the methylation site (A, B), or due to the absence of significant alternative MS/MS signals that would be present if the other glutamate of the pair were methylated (C, D). In (C) only the fragments present within the depicted region of the spectrum are indicated on the peptide sequence. A magnified view of the m/z region between 650 and 850 of (D) is shown in Fig. 6.4A. The positions of the absent alternative signals are indicated by primed fragment designations in combination with dotted arrows.
D B
C A
In the case of the Htr5 peptide DLSAAIEEVAASA only the methylation site could be unambiguously identified but not the methylation position (Fig. 3.29A). Although the peak pattern pointed to a slight preference for the first methylation position, the MS/MS spectrum also contained significant mass peaks consistent with a methylation at the alternative position. This points to the possibility that in the main fraction of the Htr5 molecules the methyl group was attached to the first Glu and in the remaining molecules it was present at the second Glu.
Figure 3.29 Examples of an unambiguous identification of the methylation site but not of the methylated glutamate residue (A), and for doubly methylated sites in a peptide derived from strain S9 (B) and in a peptide from the ∆cheB mutant WFS101 (C).
Spectra were recorded and annotated as described in Fig. 3.27. (A) Significant peaks are present at m/z values expected for an alternative methylation at the other glutamate of the pair, as indicated by primed fragment designations in combination with dotted arrows. Fig. 6.5 in the Appendix shows an MS/MS spectrum of a peptide of the same sequence but obtained from Htr8 instead of Htr5, as concluded from the gel position. In both cases the methylation position could not be determined unambiguously, but the first position seemed to be at least preferred.
C B A
The detection of a doubly methylated form of the same peptide demonstrates that in wild-type cells both residues are able to accept methyl groups (Fig. 3.29B). In the face of the detected doubly methylated peptide species, the inability to clearly identify a single methylation position indicates similar probabilities for the formation and/or hydrolysis of a methyl ester bond at both positions of this methylation site.
As mentioned, methylatable Htr peptides obtained from the ∆cheB mutant WFS101 are usually not found in the unmethylated form, but rather in singly and doubly methylated forms. An example of an unambiguous identification of such a doubly methylated peptide is shown for the Htr14 peptide DTVSATVEEIAASAN (Fig. 3.29C).