Tandem mass tag-based quantitative proteomic analysis identification of succinylation related proteins in pathogenesis of thoracic aortic aneurysm and aortic dissection

Tissue samples

The ascending aortic tissues from six thoracic aortic aneurysm (TAA) patients and six thoracic aortic dissection (TAD) patients with pre-existing aneurysms were extracted during open surgical repair. Computed tomography angiography (CTA) was performed in all patients to confirm the diagnosis. Ascending aortas from six organ donors served as controls. All patients had no family history of Marfan syndrome or other connective tissue diseases. More detailed clinical descriptions are summarized in Table 1.

All tissues were cut into approximately 1.0 × 1.0 cm² in size. Specimens were immediately frozen in liquid nitrogen and then stored in −80 °C refrigerator. The study was approved by the ethics committee of West China Hospital, Sichuan University (Sichuan, China; No. 38/2020). Written informed consent was obtained from all participants or their legal surrogates before enrolment.

Protein extraction and tryptic digestion

The tissues were carefully removed from the frozen tubes and diced into 50 mg pieces. Ten times extraction buffer was added to extract the proteins. To get rid of the tissue remnant, the extracted samples were spun.

Tissues were ground individually and lysed with ice-cold lysis buffer containing 100 mM NH₄HCO₃(pH 8), 6M Urea, and 0.2% SDS before ultrasonication on ice and centrifuged at 12,000×g for 15 min at 4 °C. For PTM detection, 3 μM trichostatin A (HY-15144; MedChemExpress, Monmouth Junction, NJ, USA), 50 mM nicotinamide (HY-B0150; MedChemExpress, Monmouth Junction, NJ, USA) and 1% phosphatase inhibitor (P1261; Solarbio, Beijing, China) were also added. BCA kit (Solarbio, Beijing, China) was used to measure the protein concentration of the supernatant. The reduction was performed with 10 mM DL-dithiothreitol for 1 h at 56 °C. Subsequently, sufficient iodoacetamide was added for 1 h alkylation in the dark at room temperature. Then samples were incubated at −20 °C for 2 h after thoroughly mixed with four times the volume of precooled acetone. Following centrifugation, the precipitation from the samples was collected, followed by dissolved in dissolution buffer (0.1M triethylammonium bicarbonate and 6M urea). Trypsin was added at 1:50 and 1:100 trypsin-to-protein mass ratios for initial overnight and second 4 h digestion, respectively. TMT 10-plex Label Reagant (Thermo Fisher Scientific, Waltham, MA, USA) was then added for 2 h at room temperature and pooled, desalted and lyophilized. Table S1 shows the TMT labeling information.

MS/MS analysis of peptides

Solvent A (2% acetonitrile, pH 10.0) was used to resuspend the peptides, and separation was performed in EASY-nLC 1200 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The liquid phase gradient follows: 94% solvent A and 6% solvent B within 2 min; 83% solvent A and 17% solvent B within 80 min; 60% solvent A and 40% solvent B within 2 min; 45% solvent A and 55% solvent B within 21 min; 100% solvent B within 5 min (solvent B: 98% acetonitrile, pH 10.0). After separation, peptides were injected into Q Exactive HF-X mass spectromer (Thermo Fisher Scientific, Waltham, MA, USA) with the following settings: spray voltage of 2.3 kV; ion transport capillary temperature of 320 °C; 350−1,500 m/z of primary scanning range; 60,000 resolution of primary MS (at m/z 200); 15,000 resolution of secondary scanning range (at m/z 200); 5 × 10⁴ target value of automatic gain control (AGC); 1.9 × 10⁵ of intensity threshold; 45 ms of maximum ion injection time and 20 s of dynamic exclusion parameter.

Data analysis

The acquired MS/MS data were analyzed against a Uniprot Homo sapiens database (2020_1_8) by Protein Discoverer (PD 2.2; Thermo Fisher Scientific, Waltham, MA, USA). Mass tolerance was set as 10 ppm and fragment mass tolerance was 0.02 Da. Carbamidomethyl on Cys was specified as fixed modifications, while oxidation Met (M) and N-terminus acetylation were set as variable modifications. TMT quantification was performed using Reporter Quantification (TMT 10-plex). False discovery rates were set as no more than 1.0% for identification of protein. Proteins containing similar peptides that indistinguishable in MS/MS analysis were identified as the same group. Mann-Whitney Test was used to statistically examine the protein quantitation results. Proteins were classified as DEPs if their quantification significantly differed between groups (fold change >1.2 or <0.83, p < 0.05).

Selection of succinylation-related proteins

We compiled a list of proteins related to succinylation according to previous published data and gene ontology (GO) terms on the AmiGO database (amigo.geneontology.org) (Bai et al., 2022; Carbon et al., 2009). The terms selected were protein succinylation, succinyl-CoA metabolic process, succinate-CoA ligase complex, succinyl-CoA:oxalate CoA-transferase, succinyl-CoA:(R)-benzylsuccinate CoA-transferase activity, succinyl-CoA binding, succinate-CoA ligase activity, succinyl-CoA hydrolase activity, succinyl-CoA:(R)-citramalate CoA-transferase activity, succinyl-CoA:3-oxo-acid CoA-transferase activity, succinate metabolic process, succinate dehydrogenase activity and succinate transport. Table S2 lists all the annotated terms and their respective GO identifications (GO ID).

Biological process enrichment analysis

Gene ontology (GO) enrichment was analyzed using the interproscan-5 program against the non-redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSiteProfiles, PANTHER). Kyoto encyclopedia of genes and genomes (KEGG) analysis was performed to analyze the pathway using Fisher’s extract test. The GO and KEGG enrichment analysis was conducted using the enrichment pipeline.

Western blotting assay

Human aortic tissues were lysed with RIPA buffer containing 3 mM trichostatin A, 50 mM nicotinamide, and 1% phosphatase inhibitor. BCA kit (Solarbio, Beijing, China) was used to measure the protein concentration of the supernatant. The lysates (30 μg) were separated by 4–20% MOPS-PAGE (ACE Biotechnology, Nanjing, China) and transferred to nitrocellulose filter membranes (Merck Millipore, Cork, IRL, USA). The membranes were blocked using 5% (w/v) nonfat dry milk in TBST Buffer, and incubated with primary antibodies against succinyllysine (PTM-401; PTM Biolabs Inc., Hangzhou, China), acetyllysine (PTM-105RM; PTM Biolabs Inc., Hangzhou, China), malonyllysine (PTM-902; PTM Biolabs Inc., Hangzhou, China), ubiquitin (PTM-1106RM; PTM Biolabs Inc., Hangzhou, China), L-lactyl lysine (PTM-1401RM; PTM Biolabs Inc., Hangzhou, China) and OXCT1 (12175-1-AP; Proteintech, Wuhan, China). Peroxidase-labeled anti-rabbit or anti-mouse IgG (ZSGB-Bio, Beijing, China) was used to probe the bound antibodies. The protein bands were visualized using enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA).

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the harvested aortic samples with TRIzol Reagent (Invitrogen, Waltham, MA, USA). Reverse transcription was carried out by PrimerScript RT reagent kit (Takara, Shiga, Japan) using 1 µg of total RNA, followed by real-time PCR analysis with SYBR Premix Ex Taq TM II (Takara, Kusatsu, Japan). The GAPDH gene was used as the endogenous control. Primer sequences were as follows: OXCT1 (Forward primer: 5′-AAGCCAAGAGAGGTGAGGGA-3′; Reverse primer: 5′-CACTGTGGTTTCTGCAGCTT-3′); GAPDH (Forward primer: 5′-GGAGCGAGATCCCTCCAAAAT-3′; Reverse primer: 5′-GGCTGTTGTCATACTTCTCATGG-3′). The relative expression of OXCT1 to GAPDH for each sample was calculated by ΔΔ Ct and expressed as 2^{–ΔΔ Ct}.

Statistical analysis

Bioinformatic analysis were performed in R software (version 4.1.1). Continuous variables were shown as mean ± standard deviation, and categorical variables were summarized as absolute numbers and percentages. Continuous variables were assessed for normality. Student’s t test was performed to make comparisons between two groups of normally distributed data, and one-way analysis of variance, followed by Tukey’s post hoc test, was used for multiple comparisons. The Mann Whitney test was performed for non-normal or non-parametric analyses. P value less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS version 24.0 (SPSS Inc., Chicago, IL, USA).

Protein lysine succinylation is high in thoracic aortic aneurysm and thoracic aortic dissection tissues

We first investigated the PTM levels including succinylation, ubiquitination, malonylation, acetylation, and lactylation in different tissues from TAA patients, TAD patients, and healthy subjects. Western blotting analysis for succinyllysine demonstrated that the levels of succinylation significantly increased in TAA and TAD patients, while others did not change remarkably (Fig. 1).

Quantitative proteomics among thoracic aortic aneurysm, thoracic aortic dissection, and normal thoracic arteries

Principal component analysis (PCA) clustergram was performed and protein profiles of all three groups have consistent clustering as shown in PCA analysis (Fig. 2A). The proteomics profiles of the TAA and TAD groups were distinctively different from those of the control group (Fig. 2B). A total of 3,293 proteins were identified from aortic aneurysm patients and healthy subjects and 3,267 proteins were identified from aortic dissection patients and healthy subjects. The differential proteins were screened with a p value < 0.05 by two-sided, unpaired t-test and a fold change >1.2 or <0.83 compared with controls.

Compared to the control group, 591 DEPs were identified in the TAA group, among which 218 proteins were upregulated and 373 proteins were downregulated; a total of 494 DEPs were identified in the TAD group, among which 212 proteins were upregulated and 282 proteins were downregulated. Among these DEPs, 197 proteins (93 upregulated and 104 downregulated) were commonly identified in TAA and TAD groups (Figs. 2C–2F). The details of these DEPs are presented in Tables S3 and S4.

Functional enrichment analysis of commonly expressed DEPs

Gene ontology analysis was performed to further clarify the underlying mechanism by biological process, cell components, and molecular function. According to the GO results, most DEPs were located in the extracellular region and participated in several functions, such as actin binding, cell adhesion molecule binding, and extracellular matrix structural constituent (Fig. 3). KEGG pathway enrichment-based clustering analysis was used to further identify the potential pathways. Most of the DEPs were mapped on “focal adhesion”, “PI3K-Akt signaling pathway” and “ECM-receptor interaction” pathways (Fig. 4).

Identification of succinylation-related proteins

Initially, a list of succinylation-related proteins was generated, as described in detail in the methods. This yielded a list of 38 related proteins (Table 2), including 10 enzymes related to succinylation modification (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, CPT1A, KAT5, and GLYATL1), 19 succinate metabolizing enzymes (SDHA, SDHB, SDHC, SDHD, SUCLA2, SUCLG1, SUCLG2, CREBBP, OXCT1, MEAF6, SSDH, SLC13A2, SLC13A3, SLC13A5, SLC16A1, SLC25A10, SLC25A11, SLC25A14, and SLC25A30) and 13 succinyl-CoA metabolizing enzymes (DLST, MMUT, ACOT4, ACOT8, NUDT7, NUDT8, NUDT19, SUCLA2, SUCLG1, SUCLG2, OGDH, OXCT1, and OXCT2). Among these succinylation-related proteins, one overlapped with the DEPs obtained as described above, namely, OXCT1 (Fig. 5A).

Evaluation of OXCT1 by Western blotting and qRT-PCR

The expression levels of OXCT1 in pathological sections were further assessed by Western blotting and qRT-PCR (Figs. 5B and 5C, Fig. S1). The results showed significantly decreased levels of OXCT1 in TAA and TAD patients relative to healthy controls (p < 0.001), confirming the differential expression patterns determined in proteomic analysis.