Identification of in vivo carbonylation sites in muscle proteins

Several bioanalytical approaches have been utilized to identify and quantify protein carbonylation. Immunochemical detection using anti-DNP antibodies in combination with 2-D gel electrophoresis was employed successfully in this work to survey protein carbonylation. Although these techniques are powerful tools to assess oxidative modifications, the actual type (i.e. HNE or aminoadipic/glutamic semialdehyde) and sites of carbonylation modification are still difficult to be determined. Mass spectrometry and proteomics-based methods, however, provide an essential framework for the rapid and accurate identification and characterization of these particular non-enzymatic post-translational modifications.

In order to identify possible carbonylation sites, the 2-D gel protein spots of several metabolic muscle enzymes, which showed the highest immunoreactivity against anti-DNP antibodies were cut out from the gel treated with trypsin and analysed by LC-tandem mass spectrometry. Tandem mass spectrometric data generated by conventional data-dependent acquisition via the LTQ-Orbitrap were extracted and searched against protein sequence database using Mascot version 2.2 search algorithm. Mascot was searched with a fragment-ion mass tolerance of 0.80 Da and a parent-ion tolerance of 5.0 ppm assuming the digestion enzyme trypsin.

Carbamidomethylation of Cys, N-terminal protein acetylation, phosphorylation of serine and threonine, aminoadipic semialdehyde/glutamic semialdehyde on Lys/Arg, HNE Michael-adduct formation on His, Lys and Cys, as well as HNE Schiff base adducts on Lys were specified as variable modifications in the database search. In general, probability-based MOWSE scores corresponding to a significance threshold of p < 0.05 were considered for peptide identification in addition to manual validation of the tandem MS data with specific emphasis on HNE-adducts identified from the database search [162].

Although the general reactivity of the nucleophilic amino acid Cys toward HNE is greater than that of His, [163] the overall higher frequency of His residues and/or inaccessibility of Cys residues under the native conditions used in the ex vivo reaction (Cys-containing peptides not modified by HNE were identified in the analysis) could account for the exclusive detection of His-HNE adducts. Moreover, a recent mass spectrometric analysis of HNE-adducts from yeast cell lysate after reaction with HNE demonstrated predominant identification of His-HNE adducts as

well [164]. In the present study the HNE adducts were identified in muscle proteins without HNE treatment prior to tandem-MS analysis, predominantly, Lys-HNE adducts in most of the cases.

This could be due to the presence of amino group Lys side chain, which provided better ionization. CID fragmentation of HNE-modified peptides may produce an intense fragment-ion signal corresponding to HNE neutral loss (-156 amu) from the precursor ion, although the intensity of the HNE neutral loss signal is not consistently the largest compared to the b- and y-type fragment ion signals across different HNE-peptide adducts. If this particular fragmentation pathway is preferred over b- and y-type ion formation as demonstrated by a predominant HNE neutral loss signal, limited sequence information or complicated spectra containing HNE neutral loss ions restrict identification of HNE modification sites, when using various database search algorithms.

Several proteins relevant to muscle function were found to be oxidatively modified including creatine kinase, adenylate kinase and triosephospate isomerase.

Creatine kinase is a metabolic muscle protein is a member of the phosphagen (guanidino) kinase family. Initially phosphocreatine was identified in muscle tissue (Eggleton & Eggleton, 1928). At that time, it was thought to be the chemical source for the energy required for muscle contraction. However, not long after, the enzyme now known as creatine kinase was first identified (Lohman, 1934), and it was subsequently shown that ATP was formed by transfer of a phosphoryl group from phosphocreatine. Analysis of the tryptic peptides by LC-tandem mass spectrometry revealed the identification of creatine kinase covering approximately 90% of the protein sequence. Very high sensitivity of LTQ Orbitrap intrument was very effective in identifying one Lys-HNE adducts and two aminoadipic semialdehyde modified peptides, which are usually present in a very low level. Figure 2.33 shows mass spectrometric characterization of the Lys-HNE modification of ¹⁵⁷LSVEALNSLTGE FK¹⁷⁰ tryptic peptide.

Figure 2.33. Positive ion nano-HPLC-ESI/MS of 4-hydroxy-2-nonenal-modified and non-modified creatine kinase tryptic peptides LSVEALNSLTGEFK from high pH muscle sample. MS spectra averaged over the chromatographic window, where peptides were eluted (51.87 min. and 54.80 respectively). (A), CID spectrum of precursor ion m/z 832.453 (2+), showing modified peptide (157-170) of full-length creatine kinase, containing the Lys-HNE modification site at K-170 with a mass shift of 156. (B), CID spectrum of precursor ion m/z 754.404 (2+), showing non-modified peptide 157-170 of full-length creatine kinase

In the series of tryptic peptides, the precursor ion at m/z 832.453 (2+) was identified as (157-170) (LSVEALNSLTGEFK) with a mass increment of 156 amu.

Modification at Lys-170 by 4-hydroxy-2-nonenal was identified by tandem-MS analysis by a complete series of yn-ions (y1-y12) in comparison to the unmodified peptide, confirming the HNE moiety at Lys-170. The most abundant species,

detected in the positive ion mode was singly charged y-ions, showing unambiguosly the location of the modified residue, while b-ions were less intense. The mass spectrum was avaraged over the chromatographic retention time in which the peptides eluted. The retention time difference between the modified peptide bearing the HNE moiety and the unmodified one was about 3 minutes. Remarkably, the modified Lys-170 residue is unaffected as a trypsin cleavage site.

Trypsin is one of the most specific proteolytic enzymes and it is most frequently used in protein structure analysis. Trypsin belongs to the serine endopeptidase family S1, which cleaves peptide bonds specifically at the C-terminal side of lysine and arginine residues. The substrate-binding pocket of trypsin has a negative charge derived from the Asp-189 residue (in bovine trypsin), which is responsible for the specific binding of the enzyme to positively charged amino acid side chains of the substrate via ionic interaction [165]. The rate of hydrolysis is substantially affected by the nature of the bond (amide or imide) between amino acid residues and by the structure of flanking sequences. Peptide bonds at the C-terminal of Arg are generally cleaved faster (2- to10-fold) than at Lys residues. The presence of acidic residues in positions of the cleavage site may lead to considerably reduced cleavage rates or even total resistance to cleavage [166].

In the series of tryptic peptides of the creatine kinase a number of Lys residues were found to be oxidized to aminoadipic semialdehyde. The lack of amino group of Lys side chain abolished the basic character of the Lys residue blocking completely the ability of trypsin to cleave the peptide bond. Figure 2.34a shows the CID-MS/MS spectrum of the double charged peptide (12-25) (LNFKAEEEYPDLSK) with the m/z 841.403. In this peptide Lys-15 was identified as being oxidized to aminoadipic semialdehyde, showed by highly intense fragment ion y11 bearing a water molecule loss. The non-modified form of this peptide is four amino acids shorter at N-terminal as the trypsin could, of course, cleave the non-modified Lys (Figure 2.34b).

Figure 2.34. (A) CID spectra of the precursor ion m/z 841.403 (2+) showing the modified peptide 12-25 of the full-length creatine kinase containing aminoadipic semialdehyde modification at K-15; (B) CID of the precursor ion 590.772 (2+) coresponding to the non-modified peptide (16-25).

LC-MS/MS analysis of the tryptic digest of creatine kinase revealed further carbonylation sites, i.e. Lys-116 (Figure 2.35). CID spectrum of the modified peptide (108-130) displayed a complete series of b and y ion at the C-terminus of the peptide No appreciable backbone fragmentation is observed at N-terminus or the peptide.

Nevertheless, Lys-116 may be regarded a possible target amino acid, because of the presence of y14-y15 and b7, b8 fragment ions.

Figure 2.35. (A) CID spectrum of the precursor ion m/z 857.738 (3+) showing the modified peptide (108-130) of the full-length creatine kinase containing aminoadipic semialdehyde modification at K-116, and, (B) CID of the precursor ion m/z 754.354 (2+) coresponding to the C-terminal unmodified peptide (117-130).

Restrictions to the specificity of trypsin occur when proline is at the carboxylic side of lysine or arginine, such bonds being highly resistant to cleavage [167]. The ring structure in proline restricts the freedom of rotation, which renders the polypeptide backbone more rigid. Sequence localization of carbonylation sites in creatine kinase identified by high performance LC-tandem MS/MS is shown in Figure 2.36. The primary amino acids sequence corresponds to creatine kinase (CK_PIG) with Swiss Prot primary accession number Q5XLD3.

1 MPFGNT NKY KLNF AEEEY PDLSKHN ALTLEIYKK LRDKETPSG FTLDDVIQTG VDNPG PFIM TVG VAGDEE SYVVFKDLFD PIIQDR GGY PTDK TDL N ENLKGGDD LDPNYVLSSR VRTG GY TLPP SRGE RRAVEKLSVE ALNSLTGEF GKYYPLKSMT EQEQQQLIDD FLFD PVSP LLLASGMARD WPDARGIW N DNKSFLVWVN EED LRVISM EKG

RRF VGLQKI EEIFKKAG P FMWNE LGYV LT PSNLGTG LRGGV VKL PKFEE ILTRLRL R GTGGVDTAAV GSVFDVSNAD RLGSSEVEQV QLVVDGV LM VEMEKKLEKG QSIDDMIPAQ K

PFGNT NKY KLNF AEEEY PDLSKHN ALTLEIYKK LRDKETPSG FTLDDVIQTG VDNPG PFIM TVG VAGDEE SYVVFKDLFD PIIQDR GGY PTDK TDL N ENLKGGDD LDPNYVLSSR VRTG GY TLPP SRGE RRAVEKLSVE ALNSLTGEF GKYYPLKSMT EQEQQQLIDD FLFD PVSP LLLASGMARD WPDARGIW N DNKSFLVWVN EED LRVISM EKG

RRF VGLQKI EEIFKKAG P FMWNE LGYV LT PSNLGTG LRGGV VKL PKFEE ILTRLRL R GTGGVDTAAV GSVFDVSNAD RLGSSEVEQV QLVVDGV LM VEMEKKLEKG QSIDDMIPAQ K

Figure 2.36. Identification of creatine kinase based on the tryptic peptide mass finger print using LC- tandem MS/MS. The obtained 90% 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), arginine, R (green), Lysine, K (black). The peptides bearing oxidative modification derived from the use of trypsin observed by LC-MS/MS are underlined and the corresponding amino acids highlighted in pink

As Figure 2.36 shows muscle creatine kinase contains 50 nucleophilic residues (4 cysteines, 15 histidines and 31 lysines). As to regards the site of HNE adduction, two parameters mainly regulate the adduction chemistry: the nucleophilic reactivity of amino acids and their accessible surface. Even though the order of reactivity of the nucleophilic amino acids towards HNE is Cys > His > Lys [168] no HNE-cysteine was identified by MS. A possible explanation could be that cysteines are involved in disulfide bonds, thus, they are not available for HNE adducts formation. Many reports have described that in vitro CK activity is inhibited by oxidative modifications elicited by exposure to oxygen radicals [169]. One possible substitute for the phosphocreatine/creatine kinase system is the adenylate kinase family proteins that catalyze the “myokinase reaction” (2ADP → ATP + AMP), which buffers ATP levels in skeletal muscles [170]. The expression of adenylate kinases in skeletal muscle fibers increases, when creatine kinase expression is relatively low [171]. Pig muscle adenylate kinase (Myokinase) is a 21.6 kDa protein and following LC-MS/MS and MASCOT database search, it was identified based on the tryptic peptides covering 93 % of the protein sequence (Figure 2.37).

1 K K C H

K

101 K K

151 K C

MEEKLKKS I IFVVGGPGSG GTQ EKIVQ KYGYT LSTG DLLRAEVSSG SARGKMLSEI MEKGQLVPLE TVLDMLRDAM VAKVDTSKGF

QGEEFERKIG QPTLLLYVDA GPETMT RLL RGETSGRVD DNEETIKKRL ETYYKATEPV IAFYE RGIV RKVNAEGSVD DVFSQV THL DTLK

51 LIDGYPREV

1 K K C H

51 K

101 K K

151 K C

MEEKLKKS I IFVVGGPGSG GTQ EKIVQ KYGYT LSTG DLLRAEVSSG SARGKMLSEI MEKGQLVPLE TVLDMLRDAM VAKVDTSKGF

QGEEFERKIG QPTLLLYVDA GPETMT RLL RGETSGRVD DNEETIKKRL ETYYKATEPV IAFYE RGIV RKVNAEGSVD DVFSQV THL DTLK

LIDGYPREV

Figure 2.37. Identification of adenylate kinase isoenzyme 1 based on the tryptic peptides mass finger print using LC-tandem MS/MS. The obtained 90% sequence coverage is shown in red. The peptides bearing oxidative modification derived from the use of trypsin observed by LC-MS/MS are underlined and the corresponding amino acids highlighted in pink

Adenylate kinase contains 21 lysine, 2 cysteines and 1 histidine residues in its sequence and a number of three were found to be oxidatively modified at lysine residues. Peptides bearing lysine oxidative modification to α-aminoadipic semialdehydes exhibit characteristic mass shifts of -1 Da. An example of lysine oxidation product is the tryptic peptide (156-167) (ATEPVIAFYEKR) presented in Figure 2.38a and b.

Figure 2.38. CID of the precursor ions of m/z 711.867 (2+) corresponding to the peptide (156-167) of the full-length adenylate kinase isoenzyme showing the formation of aminoadipic semialdehyde at K-166 by the presence of the relevant fragment ions structures (y2 and b11) of modified peptide (A), compared to the fragment ions of non-modified peptide (B).

The modification has been unequivocally assigned at Lys-166 by the complete series of y and b ions which show masses -1 Da in comparison with the unmodified fragment ions whereas b3-b10 ions are having unchanged masses.

Fragmentation of modified peptide precursor ion m/z 711.867 at MS² has led to the

formation of the main daughter ion y2 with m/z 284.1 caused by the neutral loss of H2O compared with ion y2 with m/z 303.3 in the non-modified peptide. This observation may suggest a cyclization by condensation of Lys-166 as a reason for this chemical behavior. Hence, the b11 at m/z 1230.5 fragment ion in modified peptide also exhibit water loss in comparison with b11 at m/z 1248.6 of unmodified peptide in which molecular ion exhibiting water loss is missing. In the modified lysine containing peptide the tryptic cleavage was prevented, because of the modified lysine, while in the unmodified peptide tryptic cleavage was also skipped and formally represent a missed cleavage point. According to our proposal, the loss of H2O from the protonated molecule in MS² involves the migration of γ-hydrogen from the amino group to the carboxylic oxygen group, which triggers the subsequent fragmentation.

McLafferty-type rearrangement reactions are common under soft ionization conditions, such as electrospray, leading to the abstraction of a γ-hydrogen by a group with high proton affinity, such as –OH, finally yielding H2O from alcohols, carbonyls and carbohylic acids [172].

A further example of adenylate kinase peptide sequence bearing lysine oxidation is (8-27) (SKIIFVVGGPGSGKGTQCEK) containing three lysine residues of which two of them were mass spectrometrically identified to be oxidized to α-aminoadipic semialdehyde, namely Lys-9 and Lys-21 presented in Figure 2.39. This tryptic peptide was identified in three different forms, because it was present in both modified and unmodified forms and furthermore, the tryptic cleavage was prevented in the modified forms. Figure 2.39a and b shows the formation of α-aminoadipic semialdehydes at Lys-9 and Lys-21 leading to the elongation of the corresponding tryptic peptide with one amino acid at N-terminus and six amino acids at C-terminus due to the impossibility of trypsin to cleave peptide bond at modified lysines. Figure 2.39 presents MS/MS spectra of doubly protonated tryptic peptide (SKIIFVVGGPGS GK) (m/z 672.880 2+) showing the modification site at Lys-9 by the complete series of b and y ions with the b ions having 1 Da less mass shift.

Figure 2.39. Positive ion nano-HPLC-ESI/MS showing the modification of Lys-10 and Lys-21 to aminoadipic semialdehyde (A) MS/MS product ion spectrum of [M + 2H]²⁺ ion of the precursor ions (m/z 672.880) of modified peptide (8-21) and (B) (m/z 916.964) modified peptide (10-27) (C) corresponding non-modified peptide (10-21), respectively

The second modification was identified at Lys-21 present in the tryptic peptide (10-27) (IIFVVGGPGSGKGTQCEK) m/z 916.964 (2+). The fragmentation spectrum Figure 2.39b shows incomplete set of b and y ions, but the data are sufficient to confirm the sequence of the peptide as well as the modification site. It can be observed in the series of y ions again the presence of y7-18 fragment ion at m/z 831.4 which is highly intense and the absence of y7 fragment ion. Figure 2.39c presents MS/MS data of double protonated tryptic peptide containing an unmodified lysine residue (IIFVVGGPGSGK) at m/z 565.832 (2+). However, we did not identify examples of arginine oxidation to the glutamic semialdehydes, dispite the fact that 11 arginine residues are present in the pig adenylate kinase sequence.

Further, LC-tandem mass spectrometric analysis of adenylate kinase isoenzyme 1 revealed a third carbonylation site generated by HNE Michael addition at Lys-127. The modification was identified in two different peptides, because the tryptic cleavage took place at Arg-107 and Lys-108, while HNE modified Lys-127 was preferentially cleaved by trypsin instead of Arg-128, which is the next trypsin cleavage site available toward the C-terminus. The CID fragment ions of triply charge precursor ion at m/z 783.094, show by almost complete series of y and b ions the HNE Michael addition at Lys-127 and Met-125 oxidation to methionine sulfoxide (Figure 2.40a). Lys-127 HNE modified was also found in a shorter peptide, where the Lys-108 was cleaved by trypsin (Figure 2.40b).

Figure 2.40. Positive ion nano-LC-ESI/MS of 4-hydroxy-2-nonenal-modified adenylate kinase tryptic peptides (KIGQPTLLLYVDAGPETMTK) and (IGQPTLLLYVDAGPETMTK) from high pH muscle sample. MS spectra averaged over the chromatographic window, where peptides were eluted (51.87 min.). (A) CID spectrum of precursor ion m/z 783.094 (3+), showing modified peptide (108-127) with methionine sulfoxide and HNE and (B) CID precursor ion m/z 735.064 (3+) (109-127) of full-length adenylate kinase, containing the Lys-HNE modification site at K-127 with a mass shift of 156

From the present data, it can be concluded that oxidation of lysine to aminoadipic semialdehyde block completely tryptic cleavage, whereas the formation of Lys-HNE adduct does not influence in any way trypsin activity. A very suggestiv example in this respect is the aminoadipic semialdehyde formation at Lys-49 and HNE-modification at Lys-61 identified in phosphoglycerate mutase 2 (Figure 2.41).

Figure 2.41. Nano-HPLC-tandem MS analysis of phosphoglycerate mutase 2 revealed the identification of aminoadipic semialdehyde modification at K-49, which was not cleaved by trypsin, whereas the K-61 bearing HNE moiety was cleaved by trypsin. Met-50 was identified to be modified to Met-sulfoxide in the HNE modified peptide. (A) CID of the precursor ions of m/z 917.916 (2+) aminoadipic semialdehyde modified peptide (47-61) and, (B) and 839.403 (2+) corresponding K-HNE modified peptide (50-61) of full-length phosphoglycerate mutase 2

Trypsin typically cleaves at lysine and arginine amino acid residues. The enzymatic digestion of the 4-HNE-modified protein could be retarded or even blocked by 4-HNE Schiff base or Michael additions to lysine residues on the protein, thus preventing the enzyme from recognizing possible cleavage sites. Nonetheless, previous reports showed that trypsin digestion of 4-HNE-modified cytochrome c showed 75% sequence coverage upon MALDI-TOF analysis [173]. For the first time in literature presented data shows mass spectrometric evidence that indeed HNE-modified lysine could be cleaved by trypsin, while the oxidative modification of lysine to aminoadipic semialdehyde blocks completely the tryptic cleavage.

2.4 Mass spectrometric identification of oxidative modification structures in