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.

大多数结直肠癌 （CRC） 病例由前体晚期腺瘤 （AA） 发展而来。随着蛋白质组学技术的发展，血液蛋白生物标志物在普通人群 AA 和 CRC 的早期筛查中具有潜在的应用。

AIM

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

确定用于 AA 和 CRC 早期筛查的血清蛋白生物标志物。

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.

我们从中日友好医院的 8 例正常对照 （NCs） 、19 例 AA 患者和 16 例 CRC 患者中收集了 43 份血清样本。使用液相色谱-质谱/质谱和数据非依赖性采集进行定量蛋白质组学分析，筛选出 P 值< 0.05 且绝对倍数变化> 1.5 的差异表达蛋白 （DEP），然后进行生物信息学分析。基于公共数据库进一步分析预后，并通过免疫组化验证组织中蛋白质的表达。

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.

血清样品中共鉴定出 2132 种蛋白质和 17365 种肽。NC vs AA 组有 459 个上调蛋白和 118 个下调蛋白，NC vs CRC 组有 289 个和 180 个，AA vs CRC 组有 52 个和 248 个。生物信息学分析显示，这些 DEPs 具有不同的功能，并参与广泛的信号通路。我们还发现 DIAPH1 、 VASP 、 RAB11B 、 LBP 、 SAR1A 、 TUBGCP5 和 DOK3 是 AA 和 CRC 进展的重要蛋白。此外，VASP （P < 0.01） 、LBP （P = 0.01） 、TUBGCP5 （P < 0.01） 和 DOK3 （P < 0.01） 与不良预后相关。此外，我们提出 LBP 和 VASP 可能是更有前途的蛋白质生物标志物，用于结直肠肿瘤的早期筛查。

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.

我们的研究阐明了 AA 和 CRC 患者的血清蛋白质组学特征，鉴定出的 LBP 和 VASP 等蛋白可能有助于 AA 和 CRC 的早期检测。

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

关键字：血清蛋白质组学， 晚期腺瘤， 结直肠癌， 蛋白质生物标志物， 早期筛查

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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.