Open experiment: quantitative proteomics analysis of thyroid-cancer tissue slices using ultra-high performance liquid chromatography-tandem mass spectrometry

doi:10.3724/SP.J.1123.2024.07009

Abstract

Abstract:

Open experiments play crucial roles in developing undergraduate students’ practical abilities, innovative thinking, and teamwork. This open experiment is designed to use ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in proteomics, a frontier field, by quantitatively analyzing thyroid-cancer-tissue-slice samples. Various reagents are first screened for further study by evaluating their abilities to extract proteins. This approach aims to foster students’ hands-on abilities and their scientific-research thinking. The liquid-chromatography method is subsequently optimized to enable deep-coverage of the proteome, thereby enhancing students’ understanding of liquid chromatography and mass spectrometry. Paraffin-embedded thyroid-cancer-tissue slices are finally subjected to quantitative proteomic analysis, resulting in the identification of 33 differentially expressed proteins, thereby demonstrating their potential use in disease-typing applications. This open experiment integrates theoretical and experimental knowledge gained through instrumental analysis, analytical chemistry, and biochemistry, thereby imparting scientific research thinking and innovative spirit to undergraduates. It also provides students with opportunities to expand and solidify theoretical knowledge through hands-on instrumental operation and experiments, which helps to build a systematic knowledge framework and develop a comprehensive understanding of relevant fields. Moreover, it stimulates undergraduate interest in scientific research, cultivates innovative thinking, and fosters team cooperation.

Key words: liquid chromatography (LC), mass spectrometry (MS), proteomics, label-free quantification, experimental teaching

CLC Number:

O658

LI Yilan, YUAN Huiming, CAO Jingtian, ZHENG Yidi, LI Lan, GAO Peifeng. Open experiment: quantitative proteomics analysis of thyroid-cancer tissue slices using ultra-high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2025, 43(3): 275-282.

Figures/Tables 8

Fig. 1 (a) Numbers and (b) Venn diagram of identified proteins under different protein extraction buffers GuHCl: guanidine hydrochloride; SDS: sodium dodecyl sulfate.

Fig. 2 Total ion chromatograms of peptides with different separation gradients

Fig. 3 Extracted ion chromatograms of the ion (m/z 855.46747) under different separation gradients

Fig. 4 (a) Numbers of identified proteins and peptides under different separation gradients; (b) Venn diagram of identified proteins

Fig. 5 (a) Chromatographic peaks of the ion (m/z 855.46747) under the separation gradient of 85 min, (b) Venn diagram of identified proteins and (c) Pearson correlation coefficients of the intensity within three replicated experiments R1: replicate 1; R2: replicate 2; R3: replicate 3.

Fig. 6 Repeatability of the proteins quantified from tissue slices a. goiter (G); b. thyroid papillary carcinoma T1N0M0 (T).

Fig. 7 (a) Volcano plots of proteins identified from thyroid papillary carcinoma and goiter tissue slices, and (b) unsupervised clustering of differential proteins T/G: the ratio of intensities of proteins identified in thyroid papillary carcinoma patients and goiter patients.

Fig. 8 Gene ontology (a) biological process, (b) KEGG pathway, and (c) molecular function analysis of differential proteins between goiter and thyroid papillary carcinoma tissue slices

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