Tan C, Qin G, Wang Q Q, et al. Comprehensive serum proteomics profiles and potential protein biomarkers for the early detection of advanced adenoma and colorectal cancer[J]. World Journal of Gastrointestinal Oncology, 2024, 16(7): 2971.

Comprehensive serum proteomics profiles and potential protein biomarkers for the early detection of advanced adenoma and colorectal cancer

Abstract

BACKGROUND

The majority of colorectal cancer (CRC) cases develop from precursor advanced adenoma (AA). With the development of proteomics technologies, blood protein biomarkers have potential applications in the early screening of AA and CRC in the general population.

大多數結直腸癌 （CRC） 病例由前體晚期腺瘤 （AA） 發展而來。隨著蛋白質組學技術的發展，血液蛋白生物標志物在普通人群 AA 和 CRC 的早期篩查中具有潛在的應用。

AIM

To identify serum protein biomarkers for the early screening of AA and CRC.

確定用于 AA 和 CRC 早期篩查的血清蛋白生物標志物。

METHODS

We collected 43 serum samples from 8 normal controls (NCs), 19 AA patients and 16 CRC patients at China-Japan Friendship Hospital. Quantitative proteomic analysis was performed using liquid chromatography–mass spectrometry/mass spectrometry and data independent acquisition, and differentially expressed proteins (DEPs) with P-values < 0.05 and absolute fold changes > 1.5 were screened out, followed by bioinformatics analysis. Prognosis was further analyzed based on public databases, and proteins expression in tissues were validated by immunohistochemistry.

我們從中日友好醫院的 8 例正常對照 （NCs） 、19 例 AA 患者和 16 例 CRC 患者中收集了 43 份血清樣本。使用液相色譜-質譜/質譜和數據非依賴性采集進行定量蛋白質組學分析，篩選出 P 值< 0.05 且絕對倍數變化> 1.5 的差異表達蛋白 （DEP），然后進行生物信息學分析。基于公共數據庫進一步分析預后，并通過免疫組化驗證組織中蛋白質的表達。

RESULTS

A total of 2132 proteins and 17365 peptides were identified in the serum samples. There were 459 upregulated proteins and 118 downregulated proteins in the NC vs AA group, 289 and 180 in the NC vs CRC group, and 52 and 248 in the AA vs CRC group, respectively. Bioinformatic analysis revealed that these DEPs had different functions and participated in extensive signaling pathways. We also identified DIAPH1, VASP, RAB11B, LBP, SAR1A, TUBGCP5, and DOK3 as important proteins for the progression of AA and CRC. Furthermore, VASP (P < 0.01), LBP (P = 0.01), TUBGCP5 (P < 0.01), and DOK3 (P < 0.01) were associated with a poor prognosis. In addition, we propose that LBP and VASP may be more promising protein biomarkers for the early screening of colorectal tumors.

血清樣品中共鑒定出 2132 種蛋白質和 17365 種肽。NC vs AA 組有 459 個上調蛋白和 118 個下調蛋白，NC vs CRC 組有 289 個和 180 個，AA vs CRC 組有 52 個和 248 個。生物信息學分析顯示，這些 DEPs 具有不同的功能，并參與廣泛的信號通路。我們還發現 DIAPH1 、 VASP 、 RAB11B 、 LBP 、 SAR1A 、 TUBGCP5 和 DOK3 是 AA 和 CRC 進展的重要蛋白。此外，VASP （P < 0.01） 、LBP （P = 0.01） 、TUBGCP5 （P < 0.01） 和 DOK3 （P < 0.01） 與不良預后相關。此外，我們提出 LBP 和 VASP 可能是更有前途的蛋白質生物標志物，用于結直腸腫瘤的早期篩查。

CONCLUSION

Our study elucidated the serum proteomic profiles of AA and CRC patients, and the identified proteins, such as LBP and VASP, may contribute to the early detection of AA and CRC.

我們的研究闡明了 AA 和 CRC 患者的血清蛋白質組學特征，鑒定出的 LBP 和 VASP 等蛋白可能有助于 AA 和 CRC 的早期檢測。

Keywords: Serum proteomics, Advanced adenoma, Colorectal cancer, Protein biomarker, Early screening

關鍵字：血清蛋白質組學， 晚期腺瘤， 結直腸癌， 蛋白質生物標志物， 早期篩查

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Sample collection and preparation

Fresh whole blood samples were collected from NCs, AA patients and CRC patients before surgery and centrifuged at 2-8 °C and 3000 r/min for 15 min at room temperature. The supernatant serum was collected into 2 mL freezing tubes and transferred to a -80 °C refrigerator for storage.

The sample preparation steps included low-abundance protein enrichment, protein denaturation, reduction, and alkylation as well as the digestion and peptide cleanup. Briefly, 1 mg of PuriMag magnetic beads (PuriMag Biotech, Xiamen, China) was diluted with 100 μL of wash buffer (10 mmol/L Tris (pH = 7.4), 150 mmol/L KCl, 0.05% CHAPS), and then 100 μL of serum was added. Then, the mixture was incubated at 37 °C for 1 h at 1000 rpm. After incubation, the beads were collected by the magnetic separation device and further washed with 300 μL of wash buffer three times with the magnetic separation device. The precipitate was resuspended in 40 μL of Lyse buffer (0.1 M urea, 5 mmol/L TCEP, 10 mmol/L CAA) and heated at 95 °C for 5 min at 1000 rpm with agitation. After cooling to room temperature, Lys-C and trypsin solution were added, and the sample was incubated at 37 °C for 2 h at 500 rpm with shaking. The digestion process was stopped with 10% TFA. Sample clean-up and desalting were carried out by a C18 peptide cleaning column. Peptides were eluted twice with 30 μL of elution buffer (90% ACN, 0.2% TFA) and then dried in a speed vacuum concentrator.

Proteomic signatures

We identified a total of 2132 proteins and 17365 peptides in the serum samples.