2 RESULTS and DISCUSSION
2.3 Synthesis and mass spectrometric characterization of nitrated peptides
2.3.2 High resolution mass spectrometric characterization of synthetic nitro-
Recently, high resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) has been established as a powerful tool for the mass spectrometric structure determination of protein modification, using both electrospray (ESI) and MALDI ionisation [202-204]. The feasibility and advantage of FT-ICR-MS have been demonstrated by elucidation of a specific nitration at the Tyr-430 residue of the catalytic site of bovine Prostacyclin synthase (PCS) [205].
The synthetic, nitrated and non-nitrated PCS peptides were characterised by nano-ESI-FTICR and UV/IR-MALDI-FTICR mass spectrometry as shown in Table 10.
Table 10: nano-ESI, UV-and IR-MALDI-FTICR mass spectrometric data of PCS synthetic peptides.
Nano-ESI-FTICR UV-MALDI-FTICR IR-MALDI-FTICR No. Peptide code m/z exp.
∆m
[ppm] m/z exp.
∆m
[ppm] m/z exp.
∆m [ppm]
1 PCS1 581.3043 0.17 581.3044 0.34 581.3051 1.5 2 PCS2-Y 1746.9183 0.6 1746.9188 0.86 1746.9191 1.1 3 PCS2-Y-NO2 1791.9073 2.6 191.9043 1.0 1791.9068 2.4
The nano-ESI-FTICR spectra of all Tyr-nitrated peptides showed most abundant multiply (mainly doubly and triply) protonated molecular ions without detectable fragmentation with mass determination accuracies in the low ppm range, as illustrated in Figure 63 by the peptide PCS (3), (419-432) fragment of bovine Protacyclin synthase, nitrated at the active site Tyr-430 residue. A comparative molecular modelling study of some Tyr-nitrated and non-nitrated model peptides did not indicate significant structural changes upon nitration, beyond the somewhat exposed structure of the 3-nitro group (s. insert in Figure 63).
Figure 63: nano-ESI-FTICR mass spectrum of synthetic PCS (3) peptide. The inserts show the isotopic fine structure of the [M+H]+ ion obtained by deconvolution of the most abundant triply charged ion [M+3H]3+, and a structure model of the peptide with the Tyr-430 residue using the Hyperchem III programme.
In addition, the synthetic nitro-Tyr430 PCS peptide (3) was analyzed using electrospray ionization on an Esquire 3000plus ion trap mass spectrometer. This
[M+H]+
The spectrum in Figure 64A shows exclusively the doubly, triply and quadruply charged peptide ions, indicating the molecular homogeneity of the PCS peptide. The amino acid sequence was confirmed by isolation and fragmentation of the doubly charged precursor ion of m/z 896.3 (Figure 64B). The CID mass spectrum contains the characteristic b and y fragment ions and the experimental molecular weight of 1792.12 (MWcal. = 1790.89), which are consistent with the indicated amino acid sequence PCS-(419-432). The details of the identification of b and y ions in CID spectra are presented in Appendix 4. The base peak ion in this spectrum is y132+ at m/z 838.8, indicating that the loss of the aspartic acid from the N-terminus with retention of retention of both charges on the remaining C-terminal peptide fragment represents the dominating decomposition pathway. The nitrated tyrosine was unambiguously assigned at the residue 430 of the peptide, as indicated by the fragment ion y2 (m/z 219.0) and y3 (m/z 427) which are separated by 207 amu; the shift in mass results from the presence of the nitro-group at Tyr430 (addition of 45 amu to the of a non-modified tyrosine residue). In addition, the immonium ion at m/z 180.9 is indicated for nitro-tyrosine and may be used for selective identification of nitrated tyrosine residues in complex mixture (s. Figure 64C) [107].
448.8
598.1
896.8
200 400 600 800 1000 1200 1400 1600 m/z
0.0
200 400 600 800 1000 1200 1400 1600 m/z
0.0
Figure 64: ESI-Ion trap MS of the synthetic PCS (3) peptide, (A) ESI-mass spectrum showing doubly, triply and quadruply charged peptide ions. (B) CID mass spectrum of the doubly protonated precursor ion at m/z 896.8. The observed b and y fragment ions are
170 180 190 200 210 220 230 m/z
+MS2(897.0), 9.7-10.1min
0
200 400 600 800 1000 1200 m/z
b3 / y3 b4
+MS2(897.0), 9.7-10.1min
0
200 400 600 800 1000 1200 m/z
b3 / y3 b4
The tyrosine- nitrated and non- nitrated peptides were further analysed by UV- and IR-MALDI-FT-ICR-MS, using the perpendicular UV-nitrogen at 337 nm and a 2.97µm Er: YAG infrared laser systems and identical DHB-matrix and sample preparations on the sample target of the Scout-100 MALDI source (s. Figure 65). The IR absorption, even though a factor of at least 10 weaker than those encountered in the UV, leads to enough deposition of energy into the sample to make plausible the desorption of molecules [207]. Moreover, IR-MALDI having 5-10 µm desorption depth, consumed much more material than UV-MALDI with 0.1-1µm, and only a few spectra could be obtained from the same spot in IR-MALDI analysis [93, 208].
Figure 65: Scheme of the IR-MALDI source attached to the Bruker ApexII FT-ICR- mass spectrometer equipped with the Scout 100- UV- MALDI source.
Comparable mass determination accuracies (typically 1-5 ppm) of protonated molecular ions were observed in both cases (s. Table 10). As previously reported [106] the UV-MALDI spectra showed a series of specific photochemical fragmentations for the nitrated peptides which were not observed for the unmodified peptides, with major fragment ions at [M+H-16]+, [M+H 30]+ and [M+H 32]+. This
IR Laser beam UV Laser beam
337 nm
lens lens
mirror mirror
hexapole
target
target matrix cristals
MALDI sample
(Nd-YAG): 2.97 µm
to form a nitrene-type fragment [Tyr (:N:)], and reduction of the nitro group to formally yield an amine [Tyr (NH2)] (s. Figure 66). The high mass resolution and accuracies provided by FT-ICR-MS directly ascertain the fragmentation pattern reported by Sarver et al., thus excluding other possible fragment ion types. A possible photochemical mechanism underlying this fragmentation has been previously discussed [106].
Although the UV-MALDI-induced fragmentation still provided the analysis of intact molecular ions of the synthetic 3-nitrotyrosyl- model peptides in this study, their unequivocal detection and molecular weight assignments of tyrosine nitrations in proteome analyses of proteolytic peptides mixture by low resolution UV-MALDI-MS might be significantly hampered, particularly by the possible difficulties to distinguish the [M+H -30]+ and [M+H- 32]+ ions. This problem may be further substantiated in the case of low nitration levels that are typically encountered in the study of cellular nitrated proteins [34, 205].
O
In contrast, IR-MALDI-FTICR-MS analyses of a series of 3-nitrotyrosine containing peptides provided [M+H]+ ions of high intensities without any photochemical fragmentation (Table 10, Figure 67). The stability under IR-MALDI conditions is illustrated by the spectrum of the PCS (3), (419-432) fragment of Prostacyclin synthase which showed a most abundant [M+H]+ ion at m/z 1791.9068. Using the identical DHB matrix as in UV-MALDI-FTICR-MS, the loss of water at higher laser energy was observed as the only fragmentation in IR-MALDI-FTICR spectra.
Figure 67: IR- MALDI-FT-ICR mass spectrum of PCS(3). The upper insert shows the loss of H2O at the high laser energy.
In conclusion, the ESI-MSn analysis of 3-nitrotyroeine-conatining peptides yields unambiguous results, where the introduction of the nitro group increases the molecular weight of the original peptide by + 45 atomic mass units (amu). Moreover the IR-MALDI results demonstrate the stability of 3-nitrotyrosyl peptides under
1200 1400 1600 1800 m/z
[nitro-M+H]+
[M+H]+-H2O
-18 Da
1760 1770 1780 1790 1800 1810 m/z
1791.9068
1200 1400 1600 1800 m/z
[nitro-M+H]+
[M+H]+-H2O
-18 Da
1760 1770 1780 1790 1800 1810 m/z
[M+H]+-H2O
-18 Da -18 Da
1760 1770 1780 1790 1800 1810 m/z
1791.9068
2.4 Elucidation of recognition specificity of anti 3-NT antibodies with