Mass spectrometric structural characterisation of tyrosine nitrated peptides and

Several affinity-mass spectrometry methods in combination with proteolytic digestion, have been previously developed in our laboratory [60, 62, 63, 99, 103, 104], and applied for the identification of an epitope recognition motif of an anti-nitro-tyrosine antibodies. Based on the previous study by Schmidt et al. [38], in which nitration at the Tyr-430 residue of bovine Prostacyclin synthase (PCS) near the active catalytic site was identified, several peptides comprising sequence variations at the nitration site, and nitrated peptides comprising mutated Tyr-containing peptides of PCS (Tyr-421; Tyr-83) were synthesised by solid phase peptide synthesis (SPPS). The Tyrosine and 3-Nitrotyrosine containing peptides summarised in Table 3 were synthesised on a semi-automated peptide synthesizer by SPPS using Fmoc/t-butyl protection chemistry.

Peptides with both free carboxylate and amidated C-terminal were synthesised on TGA and TGR resin, respectively.

The crude peptides released from the resin were purified by analytical and preparative RP-HPLC with the UV detection carried out at 365, 280 and 220 nm (Figure 24) and the peptides subsequently characterised by mass spectrometry.

0 10 20 30 40 50 60

0 10 20 30 40 50 60 70

Intensity(mAU)

Time (min)

365 nm

220 nm

280 nm

Figure 24: UV-RP-HPLC chromatogram of Tyr-nitrated synthetic peptide 5 at 220, 280 and 365 nm wavelengths.

HPLC characteristics and molecular ions of intact peptides obtained by MALDI-MS are summarised in Table 3. As reported earlier in studies of Tyr-nitrated peptides by UV-MALDI-MS, with the standard N2 laser operating at 337 nm wavelength, photochemical fragmentation at the nitro-group was observed as illustrated by the MALDI-FTICR- spectrum of peptide 5 (Figure 25). The specific photochemical fragmentation of the nitro-group observed in peptide 5 formally corresponds to the loss of one oxygen to form a nitroso-tyrosine derivate ([Tyr-(NO)] (A in Figure 25b), the loss of two oxygens to form a nitrene-type fragment ([Tyr-(N)] (B in Figure 25b) and the reduction of the nitro group to formally yield an amine ([Tyr-(NH₂)] (C in Figure 25b) [33, 34, 170].

Table 3: Structural characterisation and affinities of Tyr-nitrated and non-nitrated PCS peptides.

Code Sequence Nitration site

RP-HPLC Retention

time [min]

UV-MALDI-FTICR-MS^a [M+H]_exp./ [M+H]_calc.

Affinity data

Dot Blot^b 1 H-⁴¹⁶EKKDFY⁴²¹(NO₂)KDGKRL⁴²⁶-OH Y-421 28.2 1571.8124 / 1571.8182 + 2 H-⁴¹⁶EKKDFY⁴²¹KDGKRL⁴²⁶-OH - 32.5 1526.8278 / 1526.8332 - 3 H-⁷⁹DPHSY⁸³(NO₂)DAVVWEPR⁹¹-OH Y-83 27.5 1615.7073 / 1615.7142 +

4 H-⁷⁹DPHSY⁸³DAVVWEPR⁹¹-OH - 28.2 1570.7205 / 1570.7291 -

5 H-⁴²⁴GKRLKNY⁴³⁰(NO₂)SLPWGA⁴³⁶-OH Y-430 39.6 1534.8068 / 1534.8131 + 6 H-⁴²⁴GKRLKNY⁴³⁰SLPWGA⁴³⁶-OH - 32.7 1489.8204 / 1489.8280 - 7 H-⁴²⁴AKRLKNY⁴³⁰(NO2)SLPWGA⁴³⁶-OH Y-430 36.5 1548.8182 / 1548.8287 + 8 H-⁴²⁴GARLKNY⁴³⁰(NO₂)SLPWGA⁴³⁶-OH Y-430 37.2 1477.7630 / 1477.7552 + 9 H-⁴²⁴GKALKNY⁴³⁰(NO2)SLPWGA⁴³⁶-OH Y-430 38.1 1449.7487 / 1449.7491 + 10 H-⁴²⁴GKRAKNY⁴³⁰(NO2)SLPWGA⁴³⁶-OH Y-430 37.7 1492.7655 / 1492.7611 +

Code Sequence Nitration site

RP-HPLC Retention

time [min]

UV-MALDI-FTICR-MS^a [M+H]_exp./ [M+H]_calc.

Affinity data

Dot Blot^b 11 H-⁴²⁴GKRLANY⁴³⁰(NO2)SLPWGA⁴³⁶-OH Y-430 38.5 1477.7550 / 1477.7552 + 12 H-⁴²⁴GKRLKAY⁴³⁰(NO2)SLPWGA⁴³⁶-OH Y-430 38.9 1491.8123 / 1491.8073 +

13 H-⁴²⁴GKRLKASLPWGA⁴³⁶-OH - 33.6 1397.8012 / 1397.8018 -

14 H-⁴²⁷LKNY⁴³⁰(NO₂) -NH₂ Y-430 31.1 581.3123 / 581.3048 - 15 H-⁴²⁷LKNY⁴³⁰(NO₂)SLP⁴³³-OH Y-430 31.7 870.4624 / 879.4557 - 16 H-⁴²⁴GKRLKNY⁴³⁰(NO₂)SLP⁴³³-OH Y-430 32.5 1220.6831/ 1220.6752 + 17 H-⁴²¹YKDGKRLKNY⁴³⁰(NO₂)SLPWGAGHN⁴³⁶-OH Y-430 37.4 2249.132 / 2249.1216 +

a ESI-FTICR mass spectrometric measurements were carried out with a 7T Bruker Daltonics APEX III FTICR mass spectrometer incorporating an external nano-ESI ionisation source; ^b Peptide-affinities to the MAB5404 characterised by dot blot, + represents the positive response, - represents the negative response [107].

a

H-⁴²⁴GKRLKNY⁴³⁰(NO₂)SLPWGA⁴²⁴-OH

1490 1500 1510 1520 1530 1540 m/z

16 fragmentation of nitro-group in PCS peptide 5 (424-436) obtained by using an UV-MALDI-FTICR mass spectrometer with a standard N2 laser operating at 337 nm wavelength which corresponds to nitroso-Tyr derivate [Tyr-(NO)] (A), nitrene-type fragment [Tyr-(N)] (B) and amine-derivate [Tyr-(NH₂)] (C).

In contrast, the ESI-FTICR mass spectra of peptide 5 provided most abundant multiply protonated molecular ions without any detectable fragmentation, as previously described [33] (Figure 26).

[M+3H]³⁺ [M+2H]²⁺ [M+H]⁺

767.6 768.1 768.6 769.1 769.6

500 700 900 1100 1300 1500 m/z

1534.8206 512.2757 767.9104

767.9104

H-⁴²⁴GKRLKNY⁴³⁰(NO₂)SLPWGA⁴²⁴-OH

[M+H]⁺_calc: 1534.8125 [M+H]⁺_exp.: 1534.8206

∆m : 5.3 ppm

Figure 26: ESI-FTICR mass spectrum of PCS peptide 5 showing singly, doubly and triply charge ions as well as the monoisotopic fine structure of the protonated doubly charge ions.

Peptide 5 was analysed using electrospray ionisation on an ion trap mass spectrometer (Figure 27). The amino acid sequence was confirmed by isolation and fragmentation of the most abundant doubly charged precursor ion (parent ion) of m/z 767.9 in the ESI-mass spectrum. This ion was selectively trapped in the collision cell for fragmentation, and the resulting daughter ions analysed by tandem mass spectrometry.

The fragmentation pattern of m/z 767.9, mostly y and b ions, confirmed the PCS (424-436) peptide 5. The location of the nitration at Tyr-430 was determined from the y10

ion and b7 through b13 ions as illustrated in Figure 27b.

385.0

200 300 400 500 600 700 800 900 1000 1100 m/z

385.0

200 300 400 500 600 700 800 900 1000 1100 m/z

H-⁴²⁴GKRLKNY⁴³⁰(NO₂)SLPWGA⁴³⁶-OH

[M+3H]³⁺

200 400 600 800 1000 1200 1400 m/z

b₉

Figure 27: ESI-Ion trap mass spectra of the Tyr-nitrated PCS peptide 5. (a) ESI-mass spectrum showing doubly, triply and quadruply protonated charged peptide ions. (b) CID mass spectrum of the doubly protonated precursor ion at m/z 767.9. The observed b and y fragment ions are indicated in black and blue, respectively. The insert shows the Tyr-nitrated PCS peptide 5 and location of y and b ions.

A mass spectrometric structural characterisation the anti-3-nitrotyrosine antibody (MAB5404), was perfomed by an ESI-QTOF MS⁴. A desalting step of the antibody before mass spectrometric characterisation was perfomed using a C4-reverse phase column. The antibody was bound to the column, desalted with 0.1 % HCOOH, and then eluted from the column using an isocrating gradient by 55 % solvent B (95 % acetonitrile, 0.1 % HCOOH). The major ion signals were observed from m/z 2100 to m/z 3100 (Figure 28b) and main charge distribution obtained from 48+ to 70+, with the most abundant charge states being 57+, 58+ and 59+. The MassEnt1 software was applied to obtain deconvoluted spectra for the neutral mass of the intact antibody. The spectral deconvolution provides a complex pattern with multiple groups of peaks (Figure 28). The highest peak intensity at the center of the peak group indicated the most abundant mass of 149,490 Da. However the 149,490 Da peak was not homegenous and included several products differing by 2.6 kDa, which suggested some heterogeneity in the MAB5404 antibody. In the zoomed spectrum each group contained five peaks that differed by approximately 160 Da (Figure 28c), which is consistent with a previous reported difference in glycosylation [171]. Generally, an IgG molecule mass spectrum contains up to five peaks with mass difference of 162 Da due to the combination of up to four galactose losses (each heavy chain containing two galactose residues on the biantennary glycans).

63+ desalted and concentrated by using C4-reverse phase column. (a) TIC trace of LC-ESI-MS for MAB5404 antibody elution fraction; (b) ESI-MS spectrum of MAB5404 antibody; (c) Deconvoluted mass spectrum of MAB5404 antibody MaxEnt1 program; (d) “zoom” view of the deconvoluted mass spectrum of MAB5404 antibody.

The deconvoluted mass spectrum of the MAB5404 antibody showed a similar profile to an IgG1 antibody reported previously in the literature [171] (Figure 29).

149 341.0 149 491.0

149 656.0

149 803.0 150 025.0 149 173.0

149 000 149 500 150 000 150 500 mass

a b

Figure 29: Deconvoluted mass spectrum of MAB5404 antibody (a) and IgG1 antibody (b) The deconvoluted mass spectrum of the MAB5404 antibody shows a similar profile as an IgG1 antibody reported in the literature [171].

2.2.2 Binding affinity of the anti-3-nitrotyrosine antibody to tyrosine nitrated