Mass spectrometric identification of oxidative modification structures in

Besides carbonylation under the action of reactive oxygen species proteins could experience oxidative modifications, which are not carbonylation modifications.

The diversity of oxidative modification in proteins comes from the wide variety of mechanisms to induce oxidation and the propensity of specific amino acids to undergo oxidation. The most prone to oxidative attack is Cys [174] and Met [78], both containing susceptible sulfur atoms. The oxidation of Cys leads to the formation of disufide bonds, mixted disulfides and thiyl radicals [175]. The first step of methionine oxidation leads to the formation of methionine sulfoxide, while in the second step a much stronger oxidative conditions are necessary to form methionine sulfone, which is biologically irreversible. Unlike methionine oxidation, the oxidation of Cys via cysteine sulfenic acid to cysteine sulfinic acis is enzymatically reversible and possibly involved in signalling pathways [176, 177], whereas the further oxidation of cysteine to cysteic acid is irreversible and damaging [178]. The protection of cysteine against oxidation can be achieved by formation of mixted disulfides with the cysteine-containing tripeptide glutathione [179].

In addition to methionine and cysteine, tryptophan is one of the most sensitive amino acid residues to oxidizing reagents and UV light forming a number of partially isobaric compounds: hydroxytryptophan and oxindolylalanine (mass increase of 16 amu); hydroxykynurenine (+20 amu); N-formylkynurenine, dioxindolylalanine, and dihydroxytryptophan (+32 amu); kynurenine (+4 amu);

hydroxy-N-formylkynurenine (+48 amu); dihydroxy- N-formylkynurenine (164 amu);

tryptophandione and dihydrodioxoindole (+30 amu); b-unsaturated-2,4-bis-tryptophandione (+28 amu); hydroxy-bis-b-unsaturated-2,4-bis-tryptophandione (144 amu); oxolactone (+14 amu) (Figure 2.42) [180]. Recent studies revealed that Trp oxidation may represent an artefact from sample preparation using gel electrophoresis [181]. MS/MS is the only reliable technique available that can identify accordingly modified peptides and proteins, including the oxidation site due to the lack of commercial antibodies that can specifically detect oxidized tryptophan residues in proteins [182].

Figure 2.42. Possible ways for oxidation of the amino acid residues prone to oxidation by reactive oxygen species. In the first step Met is oxidized to Met sulfoxide, which may further be oxidized to Met sulfone (A), cyteine is also sulphur containing amino acid residue highly susceptible to oxidative attack forming Cys sulfenic acid, Cys sulfinic acid and cysteic acid (B). Besides Met and Cys, tryptophan oxidation leads to the formation of hydroxy-Trp (+16) and oxindolylalanine (+16). The oxidation products of oxindolyl alanine are dioxindoalanine (+32) and N-formylkynurenine (+32), which is further oxidized to hydroxyl-formylkynurenine (+48). Kynurenine (+4) is formed by decarboxylation of N-formylkynurenine, which can be oxidized to hydroxy-kynurenine (+20) (C) (Fedorova, M. et. al.)

Protein spots of beta-enolase, triosephosphate isomerase and phosphoglycerate mutase 2 were cut out from the gel, distained and digested with trypsin followed by analysis using LC-tandem mass spectrometry. Beta-enolase is a 47 kDa protein containing three tryptophan residues, which were covered in the

identified peptides. Among them only one tryptophan was found to be oxidized to kynurenine identified in the tryptic peptide ²⁸⁶NYPVVSIEDPFDQDDWK³⁰² at m/z 1035.969 (2+), which exhibited characteristic mass shifts of +4 Da (KYN) in comparison with the unmodified peptide Figure 2.43a and b.

Figure 2.43. Tandem mass spectra of tryptic beta-enolase peptide (NYPVVSIEDPFDQDDWK) displayed at m/z 1035.969 (2+) corresponding to the modified peptide to kynurenine (A) and at m/z 1033.972 (2+) of unmodified peptide (B). Comparative analysis of the CID spectra resulted from the fragmentation of both modified and unmodified precursor ions revealed the unambiguous assignement of W-301 as modification site.

A further example of kynurenine modification was identified in the tryptic peptide ¹⁶¹VVLAYEPVWAIGTGK¹⁷⁵ of triosephosphate isomerase. CID fragmentation of the precursor ion at m/z 803.944 (2+) enable identification of the modification site by the series of y7-y13 ions and b9-b14, which bear a mass shift of +4, compared with the unmodified peptide, while the y3-y6 and b3-b6 are unchanged (Figure 2.44).

Figure 2.44. MS/MS of the precursor ion of m/z 803.944 (2+) corresponding to the tryptic peptide (VVLAYEPVWAIGTGK) containing W-169 modified residue (A) and the unmodified peptide at m/z 801.949 (2+) (B). The ESI-CID spectra were recorded in the linear ion trap (LTQ) of the Orbitrap instrument for the unmodified and the modified peptide with the mass increase of +4 corresponding to the kynurenine modification

Figure 2.45 shows the MS/MS spectrum of the tryptic peptide

66TLWTILDGTDQMW PVVR⁸³ of full-length phosphogycerate mutase 2 containing three amino acid residues as possible oxidation sites. The comparative analysis of the modified and non-modified gives a mass shift of 20 amu, which theoretically corresponds to the hydroxy-kynurenine modification, but the CID fragmentation provided unambiguous structural details, showing that there are actually two amino acids modified (W-68 and Met-77) giving an overall peptide mass shift of 20 amu.

The almost complete series of y ions provided structural information that the Met-77

is oxidized to methionine sulfoxide and that the neighbouring W-78, which represents another possible oxidation site is not oxidized in any way, while the b ions show the W-68 modification to kynurenine.

Figure 2.45. Positive ion nano-HPLC-ESI/MS of kynurenine and methionine sulfoxide modified (A) and non-modified (B) tryptic peptides TLWTILDGTDQMWLPVVR of full-length phosphoglycerate mutase 2. The CID fragmentation of modified double charged precursor ion at m/z 1082.559 enable the identification of kynurenine modification at W-68 with a mass shift of +4 and methionine sulfoxide at Met-77 with 16.

Reversible post-translational modifications are usually present at moderate to low level in proteins, which make them very difficult to detect even with the most sophisticated and sensitive MS instruments such as LTQ Orbitrap. At the peptide level following enzymatic digestion MS instruments can provide access to all post-translational modifications. Besides the low level of the modifications, the limitations

may arise also from suppression effects due to: the reduced ionization efficiencies;

metal ion complexes producing several signals at lowered intensities with often unfavourable fragmentation characteristics. The coupling of RP-HPLC, which provides separation of peptide mixtures prior analysis on fast scaning instruments, inproved considerably the efficiency of post-tranlational modification analysis by MS, but even so, the weak signals of modified peptides are found often during MS analysis.

2.5 Mass spectrometric characterization of phosphorylated muscle proteins