Identification of in vivo carbonylation in α-actin

Actin is a 42 kDa contractile muscle protein, which showed a very intense immunoreactivity against anti-DNP antibody, especially in high pH (low drip loss) muscle sample. In order to characterize oxidative structure modification high performance LC-tandem MS/MS was employed as a powerful tool for unequivocal identification of carbonylation sites. Previous studies showed a significant elevation of actin carbonyl content in neurons in the brain regions most severely affected by Alzheimer’s disease pathology [157] as well as in post-ischemic isolated rat hearts [158]. Proteins mixture extracted from 48 hours high pH (low drip loss) muscle sample was separated by 2-D gel electrophoresis and the protein spot was cut out from the gel, destained and digested with trypsin. Specific carbonylation structures were identified by nano-LC-tandem MS/MS analysis of tryptic digest. Carbonyl groups are major products of reactive oxygen species-mediated oxidation reactions, arising from primary oxidative insult to protein backbone and some amino acid residues arginine, lysine, proline residues to form aminoadipic semialdehyde and glutamic semialdehyde, or 4-hydroxy-2-nonenal (HNE) a highly reactive α, β-unsaturated electrophilic aldehyde that has been shown to form Michael adducts with

cysteine, histidine, and lysine residues. Using data-dependent CID-MS the carbonylation structures were identified at the elution times between 20.2 and 60 minutes by the presence of aminoadipic semialdehydes and 4-HNE moiety. In this manner the amino acids sequences of peptides containing the oxidative modifications and their locations were elucidated. The appearance of ions corresponding to the non-modified tryptic peptides provided initial information of the peptide’s identity. In addition, a significant challenge for the “de novo” identification of carbonylated peptides and carbonylation sites was due to the primary structure of full-length muscle α-actin, which contains 17 lysines, 17 arginines, 7 histidines and 3 cysteines. All the amino acids residues susceptible for oxidation were observed in the tryptic digest of α-actin by LC-MS/MS analysis except lysine 118 and lysine 323, which yielded too short tryptic peptides.

The sequence coverage obtained by trypsin digestion of α-actin is shown in Figure 2.29. A number of three peptides with potential carbonylation sites were identified by LC-MS/MS analysis at the partial sequences (40-61), (96-113) and (313-328) (numbering as the full-length of α-actin). The CID fragmentation of these peptides was interrogated in detail to identify the sites and composition of oxidative structure.

1 DEDETTALV DNGSGLVKAG FAGDDAPRAV FPSIVGRPR QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEXGIITNWD DMEKIW TF YNELRVAPEE PTLLTEAPL NPKANREKMT QIMFETFNVP AMYVAIQAVL SLYASGRTTG IVLDSGDGVT NVPIYEGYA LP AIMRLDL AGRDLTDYLM KILTERGYSF VTTAEREIVR DIKEKL YVA LDFENEMATA ASSSSLEKSY ELPDGQVITI GNERFR PET LFQPSFIGME SAGI ETTYN R

GGTTMYPGIA DR ITAL AP I APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV RK DSYVGDEAQS KRGILTLKYP IEXGIITNWD DMEKIW TF YNELRVAPEE PTLLTEAPL NPKANREKMT QIMFETFNVP AMYVAIQAVL SLYASGRTTG IVLDSGDGVT NVPIYEGYA LP AIMRLDL AGRDLTDYLM KILTERGYSF VTTAEREIVR DIKEKL YVA LDFENEMATA ASSSSLEKSY ELPDGQVITI GNERFR PET LFQPSFIGME SAGI ETTYN R

GGTTMYPGIA DR ITAL AP I APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV RK

SIMK DIDI DLYANNVMS MQ E STM IKI

Figure 2.29. Identification of α-actin based on the tryptic peptide mass finger print using LC-tandem MS. The obtained 97% sequence coverage is shown in red. Amino acid residues that are possible sites for carbonylation are shown in different colours: cysteine, C (light blue), histidine, H (dark blue), Lysine, K (black).

A first example of peptides bearing oxidative modification derived from the use of trypsin observed by LC-MS/MS are underlined with the corresponding amino acids highlighted (in pink); LC-MS/MS provided the identification of aminoadipic modification at Lys-315 and Lys-326 assigned in two different peptides.

Figure 2.30. Positive ion nano-LC-ESI/MS/MS containing oxidized lysines in two different peptides.

Mass spectra were averaged over the chromatographic window, where the peptide was eluted (51.27and 30.92 min. respectively). (A), CID spectrum of precursor ion 782.393 (2+) of modified peptide (313-326) containing aminoadipic semialdehyde at K-315, (B), CID spectrum of precursor ion m/z 709.384 (2+), showing modified peptide (316-328) of full-length α-actin, containing oxidation site at K-326 with a mass shift of 15 amu corresponding to the methionine sulfoxide and aminoadipic semialdehyde formation. (C), Spectrum of unmodified peptide (316 –326).

Low energy collision induced dissociation tandem mass spectrometry on the LTQ Orbitrap instrument of the doubly charged precursor ion (m/z 782.393) yielded a fragment ion spectrum encompassing the amino acids residues 313-326, which contains the Lys-315 modified to aminoadipic semialdehyde. Tandem mass spectrum exhibited almost complete series of y and b ions, which enabled unambiguous identification of modification site. Noteworthy, is the observation of the highly intense fragment ion y12(-18) at m/z 1270.4 bearing water loss suggesting cyclization during CID cleavage showing the modification site. Modified lysine to aminoadipic semialdehyde blocked the trypsin action caused by the loss of basic character of lysine.

In the series of α-actin tryptic peptides the non-modified Lys-315 was cleaved by the trypsin, while Lys-326 was also found to be oxidatively modified and therefore not cleaved. The tryptic cleavage took place at the next lysine residues available located two amino acids further to the C-terminus. The tryptic peptide (316 – 328) was identified at m/z 709.38 (2+) with a mass increment of 15 amu. Isolation of the precursor ion and MS/MS provided a series of characteristic b- and y- fragment ions from which the sequence, ³¹⁶EITALAPSTMKIK³²⁸ was identified with modification Lys-326 to aminoadipic semialdehyde and at Met-325 to the sulfoxide (b), preventing cleavage by trypsin. The comparison of MS/MS spectra of modified peptide (316-328) and unmodified peptide (316- 326) cleaved after Lys-326 (b and c) provided evidence that the CID fragmentation of the peptide bond at Met-325 occurred at a higher rate for the modified peptide than for the unmodified tryptic peptide. The presence of y3 (-18) and the absence of y3 in the CID of the unmodified peptide suggested a cyclisation at Lys-326.

A further example of aminoadipic semialdehyde formation was found in the partial peptide (40-61) located at the N-terminus of the α-actin full-length sequence.

The observed triply charged precursor ion (m/z 789.350) was identified containing modification at Lys-50. Notable is the consistency and repetability of the CID fragment ions showing the modification comparing to the previous examples. In this particular case the y12(-18) was found in singly charged state at m/z 1307.3 and doubly charged state at m/z 654.5 (Figure 2.31).

Figure 2.31. Positive ion nano-HPLC-ESI/MS of α-actin tryptic peptide (40 – 61). Mass spectra were averaged over the chromatographic window, where the peptide was eluted (51.42 min.). (A), CID spectrum of precursor ion m/z 789.350 (3+), showing modified peptide (40-61) of full-length α-actin, containing oxidation site at K-50 to the aminoadipic semialdehyde formation. (B), Spectrum of unmodified peptide (51 – 61) at m/z 599.764 (2+).

Aldehydes formed by lipid peroxidation react with nucleophilic groups of proteins and are found to be selective for certain amino acid residues including cysteine (Cys), lysine (Lys), and histidine (His) [159]. The reaction between these aldehydes and amino acids occurs via Michael-type addition or Schiff base (imine) formation [160]. In HNE-based Michael-type addition, modification involves addition of the imidazole group of His (H), ɛ-amino group of Lys (K), or the sulfhydryl (–SH group of Cys (C) to the α, β-unsaturated bond of HNE [161].

Figure 2.32. Positive ion nano-LC-ESI/MS of N-hydroxynonenal-modified and non-modified α-actin tryptic peptides VAPEEHPTLLTEAPLNPK from high pH muscle sample. MS spectra averaged over the chromatographic window, where peptides were eluted (37.06 min. and 40.80, respectively). (A), CID spectrum of precursor ion m/z 704.719 (3+), showing modified peptide (96-113) of full length α-actin, containing the His-HNE modification site at His-101 with a mass shift of 156. (B), CID spectrum of precursor ion m/z 978.527 (2+), showing non-modified peptide 96-113 of full-length α-actin.

LC-MS/MS spectra of the tryptic peptide VAPEEHPTLLTEAPLNPK (residues numbers 96-113) revealed the presence of HNE adduct at His via Michael-addition (Figure 2.32). A conclusive b and y fragment ion pattern exists for a Michael adduct on His-101 of this peptide. Compared to the unmodified peptide, most notable is the 156 increase in the series of b6-b13 ions in the HNE modified peptide. The comparison of daughter y ions in the modified and unmodified peptide and the presence of b6 fragment ion in the modified peptide explained that the modification site is indeed His as no other amino acid could be candidate for HNE adduct

formation. Based upon the characteristic 156 increase in relevant fragmentation ions, it was concluded the 4-HNE reacted with the imidazole group of His-101 to form a Michael adduct.