International Journal of
Molecular Sciences Article
LC-MS/MS Analysis Unravels Deep Oxidation of Manganese Superoxide Dismutase in Kidney Cancer Zuohui Zhao 1,2 , Kazem M. Azadzoi 3 , Han-Pil Choi 3 , Ruirui Jing 1 , Xin Lu 1 , Cuiling Li 1 , Fengqin Wang 1 , Jiaju Lu 2, * and Jing-Hua Yang 1,3, * 1
2 3
*
Cancer Research Center, Shandong University School of Medicine, Jinan 250012, China; [email protected] (Z.Z.); [email protected] (R.J.); [email protected] (X.L.); [email protected] (C.L.); [email protected] (F.W.) Departments of Urology and Pediatric Surgery, Shandong Provincial Hospital Qianfoshan Hospital, Shandong University, No. 324, Jingwu Road, Jinan 250021, China Departments of Surgery, Urology, Pathology and Proteomics Laboratory, Veterans Affairs Boston Healthcare System, Boston University School of Medicine, 150 S. Huntington Ave., Boston, MA 02130, USA; [email protected] (K.M.A.); [email protected] (H.-P.C.) Correspondence: [email protected] (J.L.); [email protected] (J.-H.Y.); Tel.: +86-531-6877-6310 (J.L.); +1-857-364-5611 (J.-H.Y.); Fax: +86-531-8703-7504 (J.L.); +1-857-364-5627 (J.-H.Y.)
Academic Editor: Alan Parrish Received: 19 October 2016; Accepted: 25 January 2017; Published: 4 February 2017
Abstract: Manganese superoxide dismutase (MNSOD) is one of the major scavengers of reactive oxygen species (ROS) in mitochondria with pivotal regulatory role in ischemic disorders, inflammation and cancer. Here we report oxidative modification of MNSOD in human renal cell carcinoma (RCC) by the shotgun method using data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS). While 5816 and 5571 proteins were identified in cancer and adjacent tissues, respectively, 208 proteins were found to be up- or down-regulated (p < 0.05). Ontological category, interaction network and Western blotting suggested a close correlation between RCC-mediated proteins and oxidoreductases such as MNSOD. Markedly, oxidative modifications of MNSOD were identified at histidine (H54 and H55 ), tyrosine (Y58 ), tryptophan (W147 , W149 , W205 and W210 ) and asparagine (N206 and N209 ) residues additional to methionine. These oxidative insults were located at three hotspots near the hydrophobic pocket of the manganese binding site, of which the oxidation of Y58 , W147 and W149 was up-regulated around three folds and the oxidation of H54 and H55 was detected in the cancer tissues only (p < 0.05). When normalized to MNSOD expression levels, relative MNSOD enzymatic activity was decreased in cancer tissues, suggesting impairment of MNSOD enzymatic activity in kidney cancer due to modifications. Thus, LC-MS/MS analysis revealed multiple oxidative modifications of MNSOD at different amino acid residues that might mediate the regulation of the superoxide radicals, mitochondrial ROS scavenging and MNSOD activity in kidney cancer. Keywords: liquid chromatography-tandem mass spectrometry; MNSOD; oxidation; ROS; quantitative proteomics
1. Introduction Superoxide dismutases (SODs) are the ubiquitous superfamily of antioxidant metalloenzymes that convert the superoxide anion (O2 − ) into oxygen and hydrogen peroxide (H2 O2 ) when cells are exposed to oxidation. These enzymes are the major scavengers of reactive oxygen species (ROS), the natural side products during the process of aerobic cells respiration in physiological
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and pathological conditions [1]. Three forms of superoxide dismutases, namely Cu/ZnSOD (SOD1/3 in humans), Fe/MNSOD (also called MNSOD, SOD2 in humans), and NiSOD (only found in bacteria), are identified [2]. Among them, MNSOD is the primary mitochondrial ROS scavenging enzyme that catalyzes the conversion of superoxide to H2 O2 , which is subsequently transformed to water by catalase and other peroxidases [1,3]. MNSOD is essential for the survival of all aerobic organisms from bacteria to humans under physiological conditions [1,4]. Posttranslational modifications (PTMs) play a major role in regulating MNSOD activities, interactions, and localization [1]. Protein oxidative modification, a major class of PTM, is caused by oxidative or nitrative disruption of amino acid residues. Oxidative protein modification has been described during aging and various pathological conditions, and serves as a useful biomarker for assessing oxidative stress processes in aging and disease conditions [4]. Protein oxidation can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, sulfoxidation of methionine residues [5]. Some specific enzymes, such as tyrosine hydroxylase (TH), facilitate the oxidation process in vivo [6]. Aromatic amino acid side chains, such as tyrosine (Tyr, Y) and tryptophan (Trp, W), are more susceptible to free radical incursion leading to specific modification of the aromatic ring. For example, tyrosine (Tyr) yields greater 3,4-dihydroxyphenylalanine (DOPA) and less 2,4-isomer [7]. Protein oxidation can lead to diverse functional consequences, such as loss of enzymatic and binding activities, increased susceptibility to aggregation, proteolysis and altered immunogenicity [8]. Overall, it is believed that protein oxidation is central to functional deficit of the target proteins [9]. In addition to direct oxidative damage, cells could undergo nitrative damage through reactive nitrogen species (RNS) pathway, which often occurs with the redox-sensitive amino acid residues including tyrosine and tryptophan [10]. Nitration on tyrosine 58 (Y58 or Y34 when the 24 amino acids transit peptide is cleaved) of MNSOD has been detected by crystal structures and mutation analysis [1,11]. Previous data showed that human MNSOD Y58 was exclusively nitrated to 3-nitrotyrosine (3-NT) to inactivate its enzymatic activity, and nitration of Y58 alone was sufficient for inactivation of MNSOD enzymatic activity [1]. Similar to nitration, acetylation of MNSOD was shown to impair its enzymatic activity. Recent evidence suggests the presence of multiple MNSOD acetylation sites on lysine (Lys, K), such as 53, 68, 89, 122, 130 [12]. It was also shown that SIRT3 could deacetylate MNSOD and enhance its enzymatic activity in vitro [1,3,13]. Oxidative stress in mitochondria becomes a major source of ROS. Free radicals originating from mitochondria interact with surrounding molecules and initiate a cascade of signaling pathways leading to oxidative modifications of cellular organelles. Oxidative modifications within mitochondrial proteins may contribute to the development of carcinogenesis [14]. The precise mechanisms by which oxidative and nitrative conditions affect MNSOD structure and function remain unclear. While MNSOD is reportedly up-regulated in kidney cancer [15,16], its enzymatic activity in tumor areas is similar to adjacent kidney tissues [16,17], suggesting that MNSOD enzymatic activity does not necessarily correlate with its protein expression. It was recently shown that oxidative stress-mediated MNSOD modifications compromised its enzymatic activity and led to functional deficit [1,3,18,19]. In this study, we first pinpointed MNSOD as a vital protein in the kidney tissues from clear cell renal cell carcinoma (ccRCC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS), then attempted to identify and quantify crucial sites of oxidative modifications of MNSOD using high resolution LC-MS/MS and Progenesis LC-MS software, respectively. The newly identified (to the best of our knowledge) oxidation sites we report may introduce novel perspectives for the refinement of MNSOD regulatory mechanisms. Characterization of MNSOD oxidative modifications may be vital for interpretation of previous studies on MNSOD in tumor biology and could enhance our intervening ability in tumorigenesis.
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2.2.Results Results 2.1.Quantitative QuantitativeProteomic ProteomicAnalysis AnalysisSuggests Suggeststhe theInvolvement InvolvementofofAnti-Oxidative Anti-OxidativeStress Stress Pathway Pathway in in ccRCC ccRCC 2.1. Threepairs pairsof oftumor tumorand and adjacent adjacenttissues tissuesfrom from ccRCC ccRCCpatients patientswere were analyzed analyzed by by LC-MS/MS. LC-MS/MS. Three Totally, 5571 proteins with FDRFDR < 0.01 werewere identified in adjacent and ccRCC Totally, 5571 and and5816 5816non-redundant non-redundant proteins with < 0.01 identified in adjacent and tissues, respectively (Table S1). Among the identified proteins, the average spectral counts of three ccRCC tissues, respectively (Table S1). Among the identified proteins, the average spectral counts of repeats were used evaluate their expression levels, which revealed 100 revealed up- and 108 three repeats weretoused to evaluate their expression levels, which 100down-regulated up- and 108 proteins for at least 1.4 folds in ccRCC vs. adjacent tissues with the criteria of p < 0.05, detected down-regulated proteins for at least 1.4 folds in ccRCC vs. adjacent tissues with the criteriainofevery p< replicate, and in theevery average spectral counts of >20 (Table S2).counts According to (Table Database Annotation, 0.05, detected replicate, and the average spectral of >20 S2).for According to Visualization, and Integrated Discovery and (DAVID) functional annotation, 33 of the 208 dysregulated Database for Annotation, Visualization, Integrated Discovery (DAVID) functional annotation, proteins were related to oxido-reductases (p = 2.87 × 10−18 ) (Tables(pS3= and Heatmap 33 of the 208 dysregulated proteins were related to oxido-reductases 2.87 S4). × 10−18 ) (Tablesanalysis S3 and (Figure 1a) denoted involvement of these oxidoreductases in binding with cofactors, coenzyme, S4). Heatmap analysisthe (Figure 1a) denoted the involvement of these oxidoreductases in binding with NAD, NADH and/or NAD(P). Notably,and/or MNSODNAD(P). was among these oxidoreductases, that cofactors, coenzyme, NAD, NADH Notably, MNSOD was suggesting among these mitochondrial MNSOD was also involved in the electron transport chain for ROS removal. Consistent oxidoreductases, suggesting that mitochondrial MNSOD was also involved in the electron transport with these observations, ontological category based on biological process usingbased Search for the chain for ROS removal. Consistent with these observations, ontological category onTool biological Retrieval of Interacting Genes/Proteins (STRING) (Table S5) indicated that one of the most significant process using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (Table S5) categoriesthat was oxidation-reduction process (37 proteins, p = 6.49 × 10−17 )process (Table S6). Three main indicated one of the most significant categories was oxidation-reduction (37 proteins, p= interactive formed among the 37 interacting proteins withamong MNSOD important 6.49 × 10−17)clusters (Table were S6). Three main interactive clusters were formed theas37aninteracting node (Figure Because cancer cells usually demand1b). highBecause ROS concentrations to maintain theirhigh high proteins with 1b). MNSOD as an important node (Figure cancer cells usually demand proliferation rate [14],tothese data suggested oxido-reductases, particularly MNSOD, played an ROS concentrations maintain their highthat proliferation rate [14], these data suggested that important role in RCC pathogenesis [16].played an important role in RCC pathogenesis [16]. oxido-reductases, particularly MNSOD,
Figure 1. Cont.
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Figure 1. 1. Quantitative Quantitativeproteomic proteomicanalysis analysisrevealed revealedthe theimportance importance anti-oxidative stress pathway Figure ofof anti-oxidative stress pathway in in ccRCC. (a) The heatmap showed 33 oxidoreductases (by DAVID) were involved in cofactor, binding ccRCC. (a) The heatmap showed the 33the oxidoreductases (by DAVID) were involved in binding coenzyme, NAD, NADH or NADH NAD(P).or1:NAD(P). NAD binding site;binding 2: NADsite; or NADH binding; 3: nucleotide cofactor, coenzyme, NAD, 1: NAD 2: NAD or NADH binding; phosphate-binding region; 4: NAD(P)-binding domain; 5: NAD; 6: coenzyme 7: binding; cofactor 3: nucleotide phosphate-binding region; 4: NAD(P)-binding domain; 5: NAD; 6:binding; coenzyme binding; 8: oxidation 9: oxidoreductase. Red arrow showed candidate protein 7: cofactor binding; reduction; 8: oxidation reduction; 9: oxidoreductase. Redthe arrow showed the (MNSOD, candidate SOD2). area: SOD2). gene-term association positivelyassociation reported, light blue area: gene term protein Green (MNSOD, Green area: gene-term positively reported, lightassociation blue area: not reported yet; (b) visualization of protein–protein interactions of the 37 oxidation-reduction gene term association not reported yet; (b) visualization of protein–protein interactions ofrelated the 37 proteins in ccRCC using STRING analysis mode). 37 oxidation-reduction related proteins oxidation-reduction related proteins in (confidence ccRCC using STRING analysis (confidence mode). 37 were input into STRING software and theyinput formed mainsoftware clusters and (onlythey 33 connected proteins oxidation-reduction related proteins were intothree STRING formed three main were shown and clusters were divided dottedand lines), which red arrow) clusters (only 33the connected proteins werebyshown theamong clusters wereMNSOD divided(SOD2, by dotted lines), were participated in the(SOD2, network and werewere chosen to be validated The and solidwere lineschosen represented among which MNSOD red arrow) participated in thelater. network to be interactions between proteins thickness of the solid lines denoted the confidence levelof associated validated later. The solid linesand represented interactions between proteins and thickness the solid with interactions. lines each denoted the confidence level associated with each interactions.
2.2. 2.2. Oxidative Modification Modification of of MNSOD MNSOD For For aa deep deep post-translational post-translational modification modification analysis, analysis, MNSOD was excised from SDS-PAGE SDS-PAGE (Figure analyzed by by LC-MS/MS. LC-MS/MS.With Witha astandard standard search using MASCOT SEQUEST, (Figure 2a) and analyzed search using MASCOT andand SEQUEST, 18 18 high confident peptides MNSODwere wereidentified, identified,which whichcovered covered76% 76%of ofthe thesequence sequence (Figure (Figure 2b). high confident peptides ofof MNSOD Next, data were searched again with an open modification search algorithm [20] to Next, the the MS/MS MS/MS data to identify identify the the peptides peptides of of MNSOD MNSOD containing containing oxidation oxidation at at any any possible possible amino amino acids acids with with aa delta delta mass mass of of +16 +16 Da (Table (Table 1). 1). Totally 168 168 of of +16 +16 modification modification events were counted, counted, 81 of of which which occurred occurred at at tryptophan (W), 27 at glycine glycine (G), 24 at tyrosine tyrosine (Y), (Y), 14 14 at at histidine histidine (H), (H), 13 13 at at asparagine asparagine (N), (N), 77 at at tryptophan alanine (A), (A), and and 22 at at valine valine (V). (V). alanine
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Figure 2. 2. LC-MS/MS LC-MS/MS coverage kidney tissues tissues were were separated separated Figure coverage of of MNSOD. MNSOD. (a) (a) Whole Whole cell cell lysates lysates from from kidney by SDS-PAGE SDS-PAGE and and stained stained by by Coomassie Coomassie Blue Blue (arrow (arrow indicated indicated MNSOD). MNSOD). The The gel gel image image is is the the by representative of of 44 pairs pairs of of tumor tumor and and adjacent adjacent tissues tissues used used for for PTMs PTMs analysis. analysis. A: representative A: adjacent; adjacent; T: T: tumor; tumor; (b) CID-based CID-based sequence sequence coverage coverage of of MNSOD. MNSOD. After After Coomassie Coomassie Blue Blue staining, staining, the the 22 22 kDa kDa protein protein (b) bands corresponding to MNSOD were cut from the gel and digested, and peptides were analyzed by bands corresponding to MNSOD were cut from the gel and digested, LC-MS/MS on on LTQ-Orbitrap LTQ-Orbitrapmass massspectrometer spectrometer (MS). underlined amino letters) LC-MS/MS (MS). TheThe underlined amino acidsacids (bold(bold letters) were were identified by Proteome Discoverer 1.4 (MASCOT and SEQUEST), which covered 76.13% identified by Proteome Discoverer 1.4 (MASCOT and SEQUEST), which covered 76.13% sequence of sequence of MNSOD. Signal:peptide; the signal peptide; α: theβ:α-helices; β: thesubscript β-sheets,numbers subscriptrepresent numbers MNSOD. Signal: the signal α: the α-helices; the β-sheets, 2+ ) binding represent original solid numbers; solid arrows: (Mn2+)sites. binding sites. original numbers; arrows: metal (Mnmetal Table 1. 1. Frequency Frequency of of +16 +16 modification modification events events of of MNSOD MNSOD at at different different amino amino acid acid residues residues*.*. Table Amino Amino Acid Acid Adjacent Adjacent Tumor Tumor Total Total
A
A
3
3 44 77
C
C
0
0 00 00
D
D
0
0 00 00
E
F
G G
H
0
0 0 00 0
5 5 22 22 27 27
0 14 14 14 14
E
0 00 00
F
H 0
I
I
0
0 00 00
K
L
M
0
0 0 00 0
0 00 0
K
0 00 00
L
M
0
N
N
1
1 12 12 13 13
P
P
0
0 00 00
Q
Q
0
0 00 00
R
R
0
0 00 00
S
S
0
0 00 00
T
T
0
0 00 00
V V
0
0 22 2
W
W
28
28 53 53 81 81
Y
Y
4
4 20 20 24 24
Total
Total
41
41 127 127 168 168
* Note: counts peptidewith with a delta DaDa at the indicated amino acids.acids. * Note: totaltotal counts of of thethepeptide deltamass massofof+16 +16 at the indicated amino
To confirm whether these +16 modifications were due to oxidation, restricted search with To confirm whether these +16 modifications were due to oxidation, restricted search with dynamic dynamic oxidation (+15.995 Da) was performed using MASCOT and SEQUEST. High confident oxidation (+15.995 Da) was performed using MASCOT and SEQUEST. High confident oxidation oxidation modifications were confirmed at tryptophan (W), tyrosine (Y), histidine (H), and modifications were confirmed at tryptophan (W), tyrosine (Y), histidine (H), and asparagine (N) asparagine (N) residues of MNSOD (Table 2). Markedly, these oxidation sites were located in three residues of MNSOD (Table 2). Markedly, oxidation sites were located in three hotspots, including 55,these hotspots, including hotspot 1 (H54, H147 Y58),149hotspot 2 (W147, W149 ) and hotspot 3210 (W205, N206, N209, 54 55 58 205 206 209 hotspot 1 (H , H , Y ), hotspot 2 (W , W ) and hotspot 3 (W , N , N , W ). Additionally, W210). Additionally, the known nitration (+44.985 Da) at tyrosine (Y58) was also identified in the the known nitration (+44.985 Da) at tyrosine (Y58 ) was also identified in the kidney tissues (Table 2). kidney tissues (Table 2).
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Table 2. The modified (oxidized and nitrated) amino acid residues in MNSOD a . Amino Acid Modified
Ions Score (XCorr) d
Expectation Value
His54 His54 , His55
80 (4.9) 44 (3.7)
4.93 × 10−6 1.87 × 10−2
Trp205 , Asn206 e Asn209 e , Trp210
81 (3.8) 59 (3.9)
4.05 × 10−6 5.83 × 10−4
203 AIWNVINW(ox)ENVTER216
Trp147 Trp147 , Trp149 Trp205 Trp210
75 (5.4) 68 (4.7) 66 (4.7) 79 (5.0)
1.61 × 10−5 1.03 × 10−4 9.59 × 10−5 5.04 × 10−6
2
54 HHAAY(ox)VNNLNVTEEK68
Tyr58
96 (5.6)
1.24 × 10−7
2
54 HHAAY(no)VNNLNVTEEK68
Tyr58
27 (3.4)
4.10 × 10−1
m/z b
z
Peptide with Modification c
877.93 885.92
2 2
54 H(ox)H(ox)AAYVNNLNVTEEK68
888.44 888.44
2 2
1026.02 1034.02 880.44 880.44
2 2 2 2
877.93 892.42
54 H(ox)HAAYVNNLNVTEEK68 203 AIW(ox)N(ox)VINWENVTER216 203 AIWNVIN(ox)W(ox)ENVTER216 135 LTAASVGVQGSGW(ox)GWLGFNK154 135 LTAASVGVQGSGW(ox)GW(ox)LGFNK154 203 AIW(ox)NVINWENVTER216
a
The modified peptides in MNSOD (P04179, SODM_HUMAN), which were identified by MASCOT and SEQUEST. See Figure S1 for mass spectra containing b and y ions. b Precursor mass accuracy 38) by MASCOT, XCorr (>1.22 × charges) by SEQUEST. e Asn oxidation combined with Trp oxidation.
2.3. Tryptophan Oxidation at W147 /W149 and W205 /W210 of MNSOD Over half of the observed oxidation events of MNSOD occurred at tryptophan. Four potentially oxidized sites, W147 /W149 and W205 /W210 , were identified in two hotspots near the C-terminus. In the hotspot around the residues from 200 to 212 (hotspot 3), two of the oxidized tryptophan residues were found in the peptide AIW205 NVINW210 ENVTER. The oxidized W210 (Figure S1a) was identified by the unmodified b4–7 ions and the mass shift of the b8–13 ions, supported by the unmodified y2–6 and the modified y7–12 ions, which has been reported before [21]. Similarly, oxidation at W205 (Figure S1b) was identified by the unmodified y3–9,11 ions followed by the modified y12 ion; it was supported by the shifted b5,7–8,10–11 ions. The other oxidized tryptophan residues were found in the peptide LTAASVGVQGSGW147 GW149 LGFNK in hotspot 2. The oxidized tryptophan at W147 (Figure S1c) was uncovered by the unmodified y4–5,7 and modified y8–12,14 ions, which was supported by the unmodified b6,8–9,11 and modified b16,19 ions. Double oxidation at W147 and W149 in a different MS/MS profile of the same peptide (Figure S1d) was identified by the unmodified b6,8–9,11 ions and double mass shifts of 15.99 Da in the b15–19 ions. Markedly, these identified tryptophan oxidations at W147 , W149 , W205 and W210 were located in the hotspots that were reported to be important for the enzymatic activity of MNSOD [2]. Notably, tryptophan hydroxylase (TPH), a well characterized enzyme in mammals, was shown to convert tryptophan to 5-hydroxytryptophan, the precursor for the neurotransmitter serotonin and melatonin [22], although it was unclear whether TPH was involved in tryptophan oxidation in proteins. 2.4. Asparagine Oxidation at N206 and N209 of MNSOD The asparagine residue in proteins might be oxidized to form beta-hydroxyl asparagine [23]. We identified two asparagine oxidation sites at N206 and N209 in MNSOD, and the two oxidized asparagine residues were found in the peptide AIWN206 VIN209 WENVTER, which was located in the C-terminal. Notably, this modification was observed only in combination with other modification: double oxidation at W205 N206 and N209 W210 in the same peptide (Table 2). The oxidized N206 in the peptide (Figure S1e) was identified by the unmodified y3–10 ions and the double mass shifts of 15.99 Da in the y12 ion, and it was further supported by the double mass shifts of 15.99 Da in the ions after b4 . The oxidized N209 (Figure S1f) was identified by the first shift between the b4–6 ions and b7 ion, and the oxidized W210 identified by the second shift of 15.99 Da between the b7 ion and b8–13 ions; the double oxidized asparagine/tryptophan residues were confirmed by the unmodified y3–6 ions and double mass shifts of 15.99 Da in the y8–12 ions. Therefore, we concluded that the asparagine residues
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at N206 and N209 were hotspots for oxidation that could be spontaneously oxidized synchronously with tryptophan. 2.5. Histidine Oxidation at H54 and H55 of MNSOD Histidine oxidations in proteins are commonly found in cells undergoing redox-mediated oxidation of histidine to 2-oxo-histidine. The oxidized product has been suggested as a biological marker for oxidatively modified proteins [24]. In this study, we identified two oxidized histidine residues at H54 and H55 in MNSOD. The oxidized histidine at H54 in the peptide H(54)HAAYVNNLNVTEEK (Figure S1g) was identified by the mass shifts at b2–14 ions, which was supported by the unmodified y2–14 ions followed by the total mass shift. The double oxidized histidine at H54 and H55 in the peptide H54 H55 AAYVNNLNVTEEK (Figure S1h) was identified by double mass shifts at b11,13 ions, which was supported by the unmodified y4,6–13 ions followed by 32 Da shift of the total mass. The two oxidative histidine residues were located in the first hotspot for oxidation, which was also reported previously in human medulloblastoma cells [21]. 2.6. Tyrosine Oxidation at Y58 of MNSOD Tyrosine could be oxidized to form 3-hydroxyl tyrosine or 3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase (TH) [6,10]; however, it was unclear whether TH oxidizes the tyrosine residue in proteins. The oxidized tyrosine at Y58 was identified because the mass shift of 15.99 Da was absent in the y4–10 ions, but present in the y11–14 ions in the MS/MS profile of the peptide HHAAY58 VNNLNVTEEK (Figure S1i); it was further confirmed by the absence of the extra mass in the b2,4 ions and presence in the b5–14 ions. 2.7. Oxidation at H54 , H55 , Y58 , W147 and W149 of MNSOD Is Up-Regulated in ccRCC The peak areas were commonly considered proportional to the amounts of the peptide. In this study, the level of an oxidized residue was measured by the areas of all peptide fragments containing the oxidized residue. In comparison with unoxidized, the relative level of oxidized residues at H54 , H55 , Y58 , W147 , W149 , W205 , N206 , N209 and W210 were calculated as 0.00012, 0.00012, 0.00073, 0.09530, 0.06171, 0.06938, 0.05298, 0.05298 and 0.06938, respectively, in kidney tissues (Figure 3a, Table S7). Apparently, the basal levels of oxidation in MNSOD might indicate a managed balance between formation and neutralization of ROS in vivo. Note that abundances of W205 and N206 were identical to W210 and N209 because they were from two identical peptides with the same abundance but different pattern. The abundances of H54 and H55 were similar because they came from a low abundant, single oxidation peptide (H54 ) and a high abundant, double oxidation peptide (H54 and H55 ), so that their total abundances were mainly contributed by the high abundant peptide. Markedly, oxidation at H54 and H55 only found in kidney cancer tissues (p < 0.05, n = 4), and oxidation at Y58 , W147 and W149 increased 2.63, 3.26 and 3.13-fold (p < 0.05, n = 4) in kidney cancer tissues, respectively, in comparison with those in adjacent non-cancer tissues of the same patient (Figure 3b). While, oxidation at N206 and N209 decreased 1.54-fold (p < 0.05, n = 4) and oxidation at W205 and W210 decreased 1.40-fold (p < 0.05, n = 4) in kidney cancer tissues. Thus, these data suggested that oxidation at certain amino acid positions of MNSOD could be dysregulated in kidney cancer.
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Figure 3. 3. Quantification Quantification of of the the oxidized oxidized residues residues of of MNSOD. MNSOD. (a) (a) The The average average ratios ratios of of the the oxidized oxidized Figure to unoxidized unoxidized residues residues in in tumor tumor ++ adjacent adjacent tissues. tissues. Abundances Abundances of the the modified modified and and unmodified unmodified to 54 ,54H 55 55 147 147,,W 149, peptide fragments fragmentscontaining containingthe thenine nineoxidized oxidizedand andunoxidized unoxidizedresidues residues ,H , 58 Y58, ,WW W149 peptide (H(H ,Y 205 206 209 210 W N209 and andW W210) were ) wereused usedtotocalculate calculatethe theratios ratios(Table (Table S7). S7). Three Three hotspots are indicated; W205, N206, ,N (b) thethe oxidation modification between tumortumor and adjacent tissues. tissues. The relative of (b)comparison comparisonofof oxidation modification between and adjacent The ratios relative 54 55 58 54 55 58 oxidized to unoxidized residues at histidine 54 (H ), 55 tyrosine 58 (Y ) were bothlow in 55 (H ) and tyrosine 58 (Y low ) were ratios of oxidized to unoxidized residues at histidine 54 (H ),) and ccRCC tissues and adjacent but,tissues dramatically increased in tumor comparing with adjacent both intumor ccRCC tumor tissues andtissues adjacent but, dramatically increased in tumor comparing tissues (the lefttissues panel,(the *p< 0.03, n =*4)p (Table relative at tryptophan (W147 ) and with adjacent left panel, < 0.03, S7). n = 4)The (Table S7). ratios The relative ratios at 147 tryptophan 147 149 149)increased 149 (W also in tumor, but relative ratios at tryptophan 205 (W205 ), ) and) were 149 (W were alsosignificantly increased significantly in the tumor, but the relative ratios at tryptophan (W147 210) wereslightly asparagine (N206 ), asparagine (N209 ) and 210 (W210 ) were decreased in asparagine 206 (N206), 209 asparagine 209tryptophan (N209) and tryptophan 210 (W decreased 205 (W205), 206 tumor right panel, * p
Molecular Sciences Article
LC-MS/MS Analysis Unravels Deep Oxidation of Manganese Superoxide Dismutase in Kidney Cancer Zuohui Zhao 1,2 , Kazem M. Azadzoi 3 , Han-Pil Choi 3 , Ruirui Jing 1 , Xin Lu 1 , Cuiling Li 1 , Fengqin Wang 1 , Jiaju Lu 2, * and Jing-Hua Yang 1,3, * 1
2 3
*
Cancer Research Center, Shandong University School of Medicine, Jinan 250012, China; [email protected] (Z.Z.); [email protected] (R.J.); [email protected] (X.L.); [email protected] (C.L.); [email protected] (F.W.) Departments of Urology and Pediatric Surgery, Shandong Provincial Hospital Qianfoshan Hospital, Shandong University, No. 324, Jingwu Road, Jinan 250021, China Departments of Surgery, Urology, Pathology and Proteomics Laboratory, Veterans Affairs Boston Healthcare System, Boston University School of Medicine, 150 S. Huntington Ave., Boston, MA 02130, USA; [email protected] (K.M.A.); [email protected] (H.-P.C.) Correspondence: [email protected] (J.L.); [email protected] (J.-H.Y.); Tel.: +86-531-6877-6310 (J.L.); +1-857-364-5611 (J.-H.Y.); Fax: +86-531-8703-7504 (J.L.); +1-857-364-5627 (J.-H.Y.)
Academic Editor: Alan Parrish Received: 19 October 2016; Accepted: 25 January 2017; Published: 4 February 2017
Abstract: Manganese superoxide dismutase (MNSOD) is one of the major scavengers of reactive oxygen species (ROS) in mitochondria with pivotal regulatory role in ischemic disorders, inflammation and cancer. Here we report oxidative modification of MNSOD in human renal cell carcinoma (RCC) by the shotgun method using data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS). While 5816 and 5571 proteins were identified in cancer and adjacent tissues, respectively, 208 proteins were found to be up- or down-regulated (p < 0.05). Ontological category, interaction network and Western blotting suggested a close correlation between RCC-mediated proteins and oxidoreductases such as MNSOD. Markedly, oxidative modifications of MNSOD were identified at histidine (H54 and H55 ), tyrosine (Y58 ), tryptophan (W147 , W149 , W205 and W210 ) and asparagine (N206 and N209 ) residues additional to methionine. These oxidative insults were located at three hotspots near the hydrophobic pocket of the manganese binding site, of which the oxidation of Y58 , W147 and W149 was up-regulated around three folds and the oxidation of H54 and H55 was detected in the cancer tissues only (p < 0.05). When normalized to MNSOD expression levels, relative MNSOD enzymatic activity was decreased in cancer tissues, suggesting impairment of MNSOD enzymatic activity in kidney cancer due to modifications. Thus, LC-MS/MS analysis revealed multiple oxidative modifications of MNSOD at different amino acid residues that might mediate the regulation of the superoxide radicals, mitochondrial ROS scavenging and MNSOD activity in kidney cancer. Keywords: liquid chromatography-tandem mass spectrometry; MNSOD; oxidation; ROS; quantitative proteomics
1. Introduction Superoxide dismutases (SODs) are the ubiquitous superfamily of antioxidant metalloenzymes that convert the superoxide anion (O2 − ) into oxygen and hydrogen peroxide (H2 O2 ) when cells are exposed to oxidation. These enzymes are the major scavengers of reactive oxygen species (ROS), the natural side products during the process of aerobic cells respiration in physiological
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and pathological conditions [1]. Three forms of superoxide dismutases, namely Cu/ZnSOD (SOD1/3 in humans), Fe/MNSOD (also called MNSOD, SOD2 in humans), and NiSOD (only found in bacteria), are identified [2]. Among them, MNSOD is the primary mitochondrial ROS scavenging enzyme that catalyzes the conversion of superoxide to H2 O2 , which is subsequently transformed to water by catalase and other peroxidases [1,3]. MNSOD is essential for the survival of all aerobic organisms from bacteria to humans under physiological conditions [1,4]. Posttranslational modifications (PTMs) play a major role in regulating MNSOD activities, interactions, and localization [1]. Protein oxidative modification, a major class of PTM, is caused by oxidative or nitrative disruption of amino acid residues. Oxidative protein modification has been described during aging and various pathological conditions, and serves as a useful biomarker for assessing oxidative stress processes in aging and disease conditions [4]. Protein oxidation can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, sulfoxidation of methionine residues [5]. Some specific enzymes, such as tyrosine hydroxylase (TH), facilitate the oxidation process in vivo [6]. Aromatic amino acid side chains, such as tyrosine (Tyr, Y) and tryptophan (Trp, W), are more susceptible to free radical incursion leading to specific modification of the aromatic ring. For example, tyrosine (Tyr) yields greater 3,4-dihydroxyphenylalanine (DOPA) and less 2,4-isomer [7]. Protein oxidation can lead to diverse functional consequences, such as loss of enzymatic and binding activities, increased susceptibility to aggregation, proteolysis and altered immunogenicity [8]. Overall, it is believed that protein oxidation is central to functional deficit of the target proteins [9]. In addition to direct oxidative damage, cells could undergo nitrative damage through reactive nitrogen species (RNS) pathway, which often occurs with the redox-sensitive amino acid residues including tyrosine and tryptophan [10]. Nitration on tyrosine 58 (Y58 or Y34 when the 24 amino acids transit peptide is cleaved) of MNSOD has been detected by crystal structures and mutation analysis [1,11]. Previous data showed that human MNSOD Y58 was exclusively nitrated to 3-nitrotyrosine (3-NT) to inactivate its enzymatic activity, and nitration of Y58 alone was sufficient for inactivation of MNSOD enzymatic activity [1]. Similar to nitration, acetylation of MNSOD was shown to impair its enzymatic activity. Recent evidence suggests the presence of multiple MNSOD acetylation sites on lysine (Lys, K), such as 53, 68, 89, 122, 130 [12]. It was also shown that SIRT3 could deacetylate MNSOD and enhance its enzymatic activity in vitro [1,3,13]. Oxidative stress in mitochondria becomes a major source of ROS. Free radicals originating from mitochondria interact with surrounding molecules and initiate a cascade of signaling pathways leading to oxidative modifications of cellular organelles. Oxidative modifications within mitochondrial proteins may contribute to the development of carcinogenesis [14]. The precise mechanisms by which oxidative and nitrative conditions affect MNSOD structure and function remain unclear. While MNSOD is reportedly up-regulated in kidney cancer [15,16], its enzymatic activity in tumor areas is similar to adjacent kidney tissues [16,17], suggesting that MNSOD enzymatic activity does not necessarily correlate with its protein expression. It was recently shown that oxidative stress-mediated MNSOD modifications compromised its enzymatic activity and led to functional deficit [1,3,18,19]. In this study, we first pinpointed MNSOD as a vital protein in the kidney tissues from clear cell renal cell carcinoma (ccRCC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS), then attempted to identify and quantify crucial sites of oxidative modifications of MNSOD using high resolution LC-MS/MS and Progenesis LC-MS software, respectively. The newly identified (to the best of our knowledge) oxidation sites we report may introduce novel perspectives for the refinement of MNSOD regulatory mechanisms. Characterization of MNSOD oxidative modifications may be vital for interpretation of previous studies on MNSOD in tumor biology and could enhance our intervening ability in tumorigenesis.
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2.2.Results Results 2.1.Quantitative QuantitativeProteomic ProteomicAnalysis AnalysisSuggests Suggeststhe theInvolvement InvolvementofofAnti-Oxidative Anti-OxidativeStress Stress Pathway Pathway in in ccRCC ccRCC 2.1. Threepairs pairsof oftumor tumorand and adjacent adjacenttissues tissuesfrom from ccRCC ccRCCpatients patientswere were analyzed analyzed by by LC-MS/MS. LC-MS/MS. Three Totally, 5571 proteins with FDRFDR < 0.01 werewere identified in adjacent and ccRCC Totally, 5571 and and5816 5816non-redundant non-redundant proteins with < 0.01 identified in adjacent and tissues, respectively (Table S1). Among the identified proteins, the average spectral counts of three ccRCC tissues, respectively (Table S1). Among the identified proteins, the average spectral counts of repeats were used evaluate their expression levels, which revealed 100 revealed up- and 108 three repeats weretoused to evaluate their expression levels, which 100down-regulated up- and 108 proteins for at least 1.4 folds in ccRCC vs. adjacent tissues with the criteria of p < 0.05, detected down-regulated proteins for at least 1.4 folds in ccRCC vs. adjacent tissues with the criteriainofevery p< replicate, and in theevery average spectral counts of >20 (Table S2).counts According to (Table Database Annotation, 0.05, detected replicate, and the average spectral of >20 S2).for According to Visualization, and Integrated Discovery and (DAVID) functional annotation, 33 of the 208 dysregulated Database for Annotation, Visualization, Integrated Discovery (DAVID) functional annotation, proteins were related to oxido-reductases (p = 2.87 × 10−18 ) (Tables(pS3= and Heatmap 33 of the 208 dysregulated proteins were related to oxido-reductases 2.87 S4). × 10−18 ) (Tablesanalysis S3 and (Figure 1a) denoted involvement of these oxidoreductases in binding with cofactors, coenzyme, S4). Heatmap analysisthe (Figure 1a) denoted the involvement of these oxidoreductases in binding with NAD, NADH and/or NAD(P). Notably,and/or MNSODNAD(P). was among these oxidoreductases, that cofactors, coenzyme, NAD, NADH Notably, MNSOD was suggesting among these mitochondrial MNSOD was also involved in the electron transport chain for ROS removal. Consistent oxidoreductases, suggesting that mitochondrial MNSOD was also involved in the electron transport with these observations, ontological category based on biological process usingbased Search for the chain for ROS removal. Consistent with these observations, ontological category onTool biological Retrieval of Interacting Genes/Proteins (STRING) (Table S5) indicated that one of the most significant process using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (Table S5) categoriesthat was oxidation-reduction process (37 proteins, p = 6.49 × 10−17 )process (Table S6). Three main indicated one of the most significant categories was oxidation-reduction (37 proteins, p= interactive formed among the 37 interacting proteins withamong MNSOD important 6.49 × 10−17)clusters (Table were S6). Three main interactive clusters were formed theas37aninteracting node (Figure Because cancer cells usually demand1b). highBecause ROS concentrations to maintain theirhigh high proteins with 1b). MNSOD as an important node (Figure cancer cells usually demand proliferation rate [14],tothese data suggested oxido-reductases, particularly MNSOD, played an ROS concentrations maintain their highthat proliferation rate [14], these data suggested that important role in RCC pathogenesis [16].played an important role in RCC pathogenesis [16]. oxido-reductases, particularly MNSOD,
Figure 1. Cont.
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Figure 1. 1. Quantitative Quantitativeproteomic proteomicanalysis analysisrevealed revealedthe theimportance importance anti-oxidative stress pathway Figure ofof anti-oxidative stress pathway in in ccRCC. (a) The heatmap showed 33 oxidoreductases (by DAVID) were involved in cofactor, binding ccRCC. (a) The heatmap showed the 33the oxidoreductases (by DAVID) were involved in binding coenzyme, NAD, NADH or NADH NAD(P).or1:NAD(P). NAD binding site;binding 2: NADsite; or NADH binding; 3: nucleotide cofactor, coenzyme, NAD, 1: NAD 2: NAD or NADH binding; phosphate-binding region; 4: NAD(P)-binding domain; 5: NAD; 6: coenzyme 7: binding; cofactor 3: nucleotide phosphate-binding region; 4: NAD(P)-binding domain; 5: NAD; 6:binding; coenzyme binding; 8: oxidation 9: oxidoreductase. Red arrow showed candidate protein 7: cofactor binding; reduction; 8: oxidation reduction; 9: oxidoreductase. Redthe arrow showed the (MNSOD, candidate SOD2). area: SOD2). gene-term association positivelyassociation reported, light blue area: gene term protein Green (MNSOD, Green area: gene-term positively reported, lightassociation blue area: not reported yet; (b) visualization of protein–protein interactions of the 37 oxidation-reduction gene term association not reported yet; (b) visualization of protein–protein interactions ofrelated the 37 proteins in ccRCC using STRING analysis mode). 37 oxidation-reduction related proteins oxidation-reduction related proteins in (confidence ccRCC using STRING analysis (confidence mode). 37 were input into STRING software and theyinput formed mainsoftware clusters and (onlythey 33 connected proteins oxidation-reduction related proteins were intothree STRING formed three main were shown and clusters were divided dottedand lines), which red arrow) clusters (only 33the connected proteins werebyshown theamong clusters wereMNSOD divided(SOD2, by dotted lines), were participated in the(SOD2, network and werewere chosen to be validated The and solidwere lineschosen represented among which MNSOD red arrow) participated in thelater. network to be interactions between proteins thickness of the solid lines denoted the confidence levelof associated validated later. The solid linesand represented interactions between proteins and thickness the solid with interactions. lines each denoted the confidence level associated with each interactions.
2.2. 2.2. Oxidative Modification Modification of of MNSOD MNSOD For For aa deep deep post-translational post-translational modification modification analysis, analysis, MNSOD was excised from SDS-PAGE SDS-PAGE (Figure analyzed by by LC-MS/MS. LC-MS/MS.With Witha astandard standard search using MASCOT SEQUEST, (Figure 2a) and analyzed search using MASCOT andand SEQUEST, 18 18 high confident peptides MNSODwere wereidentified, identified,which whichcovered covered76% 76%of ofthe thesequence sequence (Figure (Figure 2b). high confident peptides ofof MNSOD Next, data were searched again with an open modification search algorithm [20] to Next, the the MS/MS MS/MS data to identify identify the the peptides peptides of of MNSOD MNSOD containing containing oxidation oxidation at at any any possible possible amino amino acids acids with with aa delta delta mass mass of of +16 +16 Da (Table (Table 1). 1). Totally 168 168 of of +16 +16 modification modification events were counted, counted, 81 of of which which occurred occurred at at tryptophan (W), 27 at glycine glycine (G), 24 at tyrosine tyrosine (Y), (Y), 14 14 at at histidine histidine (H), (H), 13 13 at at asparagine asparagine (N), (N), 77 at at tryptophan alanine (A), (A), and and 22 at at valine valine (V). (V). alanine
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Figure 2. 2. LC-MS/MS LC-MS/MS coverage kidney tissues tissues were were separated separated Figure coverage of of MNSOD. MNSOD. (a) (a) Whole Whole cell cell lysates lysates from from kidney by SDS-PAGE SDS-PAGE and and stained stained by by Coomassie Coomassie Blue Blue (arrow (arrow indicated indicated MNSOD). MNSOD). The The gel gel image image is is the the by representative of of 44 pairs pairs of of tumor tumor and and adjacent adjacent tissues tissues used used for for PTMs PTMs analysis. analysis. A: representative A: adjacent; adjacent; T: T: tumor; tumor; (b) CID-based CID-based sequence sequence coverage coverage of of MNSOD. MNSOD. After After Coomassie Coomassie Blue Blue staining, staining, the the 22 22 kDa kDa protein protein (b) bands corresponding to MNSOD were cut from the gel and digested, and peptides were analyzed by bands corresponding to MNSOD were cut from the gel and digested, LC-MS/MS on on LTQ-Orbitrap LTQ-Orbitrapmass massspectrometer spectrometer (MS). underlined amino letters) LC-MS/MS (MS). TheThe underlined amino acidsacids (bold(bold letters) were were identified by Proteome Discoverer 1.4 (MASCOT and SEQUEST), which covered 76.13% identified by Proteome Discoverer 1.4 (MASCOT and SEQUEST), which covered 76.13% sequence of sequence of MNSOD. Signal:peptide; the signal peptide; α: theβ:α-helices; β: thesubscript β-sheets,numbers subscriptrepresent numbers MNSOD. Signal: the signal α: the α-helices; the β-sheets, 2+ ) binding represent original solid numbers; solid arrows: (Mn2+)sites. binding sites. original numbers; arrows: metal (Mnmetal Table 1. 1. Frequency Frequency of of +16 +16 modification modification events events of of MNSOD MNSOD at at different different amino amino acid acid residues residues*.*. Table Amino Amino Acid Acid Adjacent Adjacent Tumor Tumor Total Total
A
A
3
3 44 77
C
C
0
0 00 00
D
D
0
0 00 00
E
F
G G
H
0
0 0 00 0
5 5 22 22 27 27
0 14 14 14 14
E
0 00 00
F
H 0
I
I
0
0 00 00
K
L
M
0
0 0 00 0
0 00 0
K
0 00 00
L
M
0
N
N
1
1 12 12 13 13
P
P
0
0 00 00
Q
Q
0
0 00 00
R
R
0
0 00 00
S
S
0
0 00 00
T
T
0
0 00 00
V V
0
0 22 2
W
W
28
28 53 53 81 81
Y
Y
4
4 20 20 24 24
Total
Total
41
41 127 127 168 168
* Note: counts peptidewith with a delta DaDa at the indicated amino acids.acids. * Note: totaltotal counts of of thethepeptide deltamass massofof+16 +16 at the indicated amino
To confirm whether these +16 modifications were due to oxidation, restricted search with To confirm whether these +16 modifications were due to oxidation, restricted search with dynamic dynamic oxidation (+15.995 Da) was performed using MASCOT and SEQUEST. High confident oxidation (+15.995 Da) was performed using MASCOT and SEQUEST. High confident oxidation oxidation modifications were confirmed at tryptophan (W), tyrosine (Y), histidine (H), and modifications were confirmed at tryptophan (W), tyrosine (Y), histidine (H), and asparagine (N) asparagine (N) residues of MNSOD (Table 2). Markedly, these oxidation sites were located in three residues of MNSOD (Table 2). Markedly, oxidation sites were located in three hotspots, including 55,these hotspots, including hotspot 1 (H54, H147 Y58),149hotspot 2 (W147, W149 ) and hotspot 3210 (W205, N206, N209, 54 55 58 205 206 209 hotspot 1 (H , H , Y ), hotspot 2 (W , W ) and hotspot 3 (W , N , N , W ). Additionally, W210). Additionally, the known nitration (+44.985 Da) at tyrosine (Y58) was also identified in the the known nitration (+44.985 Da) at tyrosine (Y58 ) was also identified in the kidney tissues (Table 2). kidney tissues (Table 2).
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Table 2. The modified (oxidized and nitrated) amino acid residues in MNSOD a . Amino Acid Modified
Ions Score (XCorr) d
Expectation Value
His54 His54 , His55
80 (4.9) 44 (3.7)
4.93 × 10−6 1.87 × 10−2
Trp205 , Asn206 e Asn209 e , Trp210
81 (3.8) 59 (3.9)
4.05 × 10−6 5.83 × 10−4
203 AIWNVINW(ox)ENVTER216
Trp147 Trp147 , Trp149 Trp205 Trp210
75 (5.4) 68 (4.7) 66 (4.7) 79 (5.0)
1.61 × 10−5 1.03 × 10−4 9.59 × 10−5 5.04 × 10−6
2
54 HHAAY(ox)VNNLNVTEEK68
Tyr58
96 (5.6)
1.24 × 10−7
2
54 HHAAY(no)VNNLNVTEEK68
Tyr58
27 (3.4)
4.10 × 10−1
m/z b
z
Peptide with Modification c
877.93 885.92
2 2
54 H(ox)H(ox)AAYVNNLNVTEEK68
888.44 888.44
2 2
1026.02 1034.02 880.44 880.44
2 2 2 2
877.93 892.42
54 H(ox)HAAYVNNLNVTEEK68 203 AIW(ox)N(ox)VINWENVTER216 203 AIWNVIN(ox)W(ox)ENVTER216 135 LTAASVGVQGSGW(ox)GWLGFNK154 135 LTAASVGVQGSGW(ox)GW(ox)LGFNK154 203 AIW(ox)NVINWENVTER216
a
The modified peptides in MNSOD (P04179, SODM_HUMAN), which were identified by MASCOT and SEQUEST. See Figure S1 for mass spectra containing b and y ions. b Precursor mass accuracy 38) by MASCOT, XCorr (>1.22 × charges) by SEQUEST. e Asn oxidation combined with Trp oxidation.
2.3. Tryptophan Oxidation at W147 /W149 and W205 /W210 of MNSOD Over half of the observed oxidation events of MNSOD occurred at tryptophan. Four potentially oxidized sites, W147 /W149 and W205 /W210 , were identified in two hotspots near the C-terminus. In the hotspot around the residues from 200 to 212 (hotspot 3), two of the oxidized tryptophan residues were found in the peptide AIW205 NVINW210 ENVTER. The oxidized W210 (Figure S1a) was identified by the unmodified b4–7 ions and the mass shift of the b8–13 ions, supported by the unmodified y2–6 and the modified y7–12 ions, which has been reported before [21]. Similarly, oxidation at W205 (Figure S1b) was identified by the unmodified y3–9,11 ions followed by the modified y12 ion; it was supported by the shifted b5,7–8,10–11 ions. The other oxidized tryptophan residues were found in the peptide LTAASVGVQGSGW147 GW149 LGFNK in hotspot 2. The oxidized tryptophan at W147 (Figure S1c) was uncovered by the unmodified y4–5,7 and modified y8–12,14 ions, which was supported by the unmodified b6,8–9,11 and modified b16,19 ions. Double oxidation at W147 and W149 in a different MS/MS profile of the same peptide (Figure S1d) was identified by the unmodified b6,8–9,11 ions and double mass shifts of 15.99 Da in the b15–19 ions. Markedly, these identified tryptophan oxidations at W147 , W149 , W205 and W210 were located in the hotspots that were reported to be important for the enzymatic activity of MNSOD [2]. Notably, tryptophan hydroxylase (TPH), a well characterized enzyme in mammals, was shown to convert tryptophan to 5-hydroxytryptophan, the precursor for the neurotransmitter serotonin and melatonin [22], although it was unclear whether TPH was involved in tryptophan oxidation in proteins. 2.4. Asparagine Oxidation at N206 and N209 of MNSOD The asparagine residue in proteins might be oxidized to form beta-hydroxyl asparagine [23]. We identified two asparagine oxidation sites at N206 and N209 in MNSOD, and the two oxidized asparagine residues were found in the peptide AIWN206 VIN209 WENVTER, which was located in the C-terminal. Notably, this modification was observed only in combination with other modification: double oxidation at W205 N206 and N209 W210 in the same peptide (Table 2). The oxidized N206 in the peptide (Figure S1e) was identified by the unmodified y3–10 ions and the double mass shifts of 15.99 Da in the y12 ion, and it was further supported by the double mass shifts of 15.99 Da in the ions after b4 . The oxidized N209 (Figure S1f) was identified by the first shift between the b4–6 ions and b7 ion, and the oxidized W210 identified by the second shift of 15.99 Da between the b7 ion and b8–13 ions; the double oxidized asparagine/tryptophan residues were confirmed by the unmodified y3–6 ions and double mass shifts of 15.99 Da in the y8–12 ions. Therefore, we concluded that the asparagine residues
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at N206 and N209 were hotspots for oxidation that could be spontaneously oxidized synchronously with tryptophan. 2.5. Histidine Oxidation at H54 and H55 of MNSOD Histidine oxidations in proteins are commonly found in cells undergoing redox-mediated oxidation of histidine to 2-oxo-histidine. The oxidized product has been suggested as a biological marker for oxidatively modified proteins [24]. In this study, we identified two oxidized histidine residues at H54 and H55 in MNSOD. The oxidized histidine at H54 in the peptide H(54)HAAYVNNLNVTEEK (Figure S1g) was identified by the mass shifts at b2–14 ions, which was supported by the unmodified y2–14 ions followed by the total mass shift. The double oxidized histidine at H54 and H55 in the peptide H54 H55 AAYVNNLNVTEEK (Figure S1h) was identified by double mass shifts at b11,13 ions, which was supported by the unmodified y4,6–13 ions followed by 32 Da shift of the total mass. The two oxidative histidine residues were located in the first hotspot for oxidation, which was also reported previously in human medulloblastoma cells [21]. 2.6. Tyrosine Oxidation at Y58 of MNSOD Tyrosine could be oxidized to form 3-hydroxyl tyrosine or 3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase (TH) [6,10]; however, it was unclear whether TH oxidizes the tyrosine residue in proteins. The oxidized tyrosine at Y58 was identified because the mass shift of 15.99 Da was absent in the y4–10 ions, but present in the y11–14 ions in the MS/MS profile of the peptide HHAAY58 VNNLNVTEEK (Figure S1i); it was further confirmed by the absence of the extra mass in the b2,4 ions and presence in the b5–14 ions. 2.7. Oxidation at H54 , H55 , Y58 , W147 and W149 of MNSOD Is Up-Regulated in ccRCC The peak areas were commonly considered proportional to the amounts of the peptide. In this study, the level of an oxidized residue was measured by the areas of all peptide fragments containing the oxidized residue. In comparison with unoxidized, the relative level of oxidized residues at H54 , H55 , Y58 , W147 , W149 , W205 , N206 , N209 and W210 were calculated as 0.00012, 0.00012, 0.00073, 0.09530, 0.06171, 0.06938, 0.05298, 0.05298 and 0.06938, respectively, in kidney tissues (Figure 3a, Table S7). Apparently, the basal levels of oxidation in MNSOD might indicate a managed balance between formation and neutralization of ROS in vivo. Note that abundances of W205 and N206 were identical to W210 and N209 because they were from two identical peptides with the same abundance but different pattern. The abundances of H54 and H55 were similar because they came from a low abundant, single oxidation peptide (H54 ) and a high abundant, double oxidation peptide (H54 and H55 ), so that their total abundances were mainly contributed by the high abundant peptide. Markedly, oxidation at H54 and H55 only found in kidney cancer tissues (p < 0.05, n = 4), and oxidation at Y58 , W147 and W149 increased 2.63, 3.26 and 3.13-fold (p < 0.05, n = 4) in kidney cancer tissues, respectively, in comparison with those in adjacent non-cancer tissues of the same patient (Figure 3b). While, oxidation at N206 and N209 decreased 1.54-fold (p < 0.05, n = 4) and oxidation at W205 and W210 decreased 1.40-fold (p < 0.05, n = 4) in kidney cancer tissues. Thus, these data suggested that oxidation at certain amino acid positions of MNSOD could be dysregulated in kidney cancer.
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Figure 3. 3. Quantification Quantification of of the the oxidized oxidized residues residues of of MNSOD. MNSOD. (a) (a) The The average average ratios ratios of of the the oxidized oxidized Figure to unoxidized unoxidized residues residues in in tumor tumor ++ adjacent adjacent tissues. tissues. Abundances Abundances of the the modified modified and and unmodified unmodified to 54 ,54H 55 55 147 147,,W 149, peptide fragments fragmentscontaining containingthe thenine nineoxidized oxidizedand andunoxidized unoxidizedresidues residues ,H , 58 Y58, ,WW W149 peptide (H(H ,Y 205 206 209 210 W N209 and andW W210) were ) wereused usedtotocalculate calculatethe theratios ratios(Table (Table S7). S7). Three Three hotspots are indicated; W205, N206, ,N (b) thethe oxidation modification between tumortumor and adjacent tissues. tissues. The relative of (b)comparison comparisonofof oxidation modification between and adjacent The ratios relative 54 55 58 54 55 58 oxidized to unoxidized residues at histidine 54 (H ), 55 tyrosine 58 (Y ) were bothlow in 55 (H ) and tyrosine 58 (Y low ) were ratios of oxidized to unoxidized residues at histidine 54 (H ),) and ccRCC tissues and adjacent but,tissues dramatically increased in tumor comparing with adjacent both intumor ccRCC tumor tissues andtissues adjacent but, dramatically increased in tumor comparing tissues (the lefttissues panel,(the *p< 0.03, n =*4)p (Table relative at tryptophan (W147 ) and with adjacent left panel, < 0.03, S7). n = 4)The (Table S7). ratios The relative ratios at 147 tryptophan 147 149 149)increased 149 (W also in tumor, but relative ratios at tryptophan 205 (W205 ), ) and) were 149 (W were alsosignificantly increased significantly in the tumor, but the relative ratios at tryptophan (W147 210) wereslightly asparagine (N206 ), asparagine (N209 ) and 210 (W210 ) were decreased in asparagine 206 (N206), 209 asparagine 209tryptophan (N209) and tryptophan 210 (W decreased 205 (W205), 206 tumor right panel, * p
