LC-MS/MS analysis of human thyroid cystic fluid and thyroid cancer cell secreted proteins

Thyroid ultrasound and ultrasound-guided fine-needle aspiration (USG/FNA) biopsy are currently used for diagnosing papillary thyroid carcinoma (PTC), but their detection limit could be improved by combining other biomarkers. To discover novel PTC biomarkers, we herein applied a GeLC-MS/MS strategy to analyze the proteome profiles of serum-abundant-protein-depleted FNA cystic fluid from benign and PTC patients, as well as two PTC cell line secretomes. From them, we identified 346, 488, and 2105 proteins, respectively. Comparative analysis revealed that 191 proteins were detected in the PTC but not the benign cystic fluid samples, and thus may represent potential PTC biomarkers. Among these proteins, 101 were detected in the PTC cell line secretomes, and seven of them (NPC2, CTSC, AGRN, GPNMB, DPP4, ERAP2, and SH3BGRL3) were reported in public PTC transcriptome datasets as having 4681 elevated mRNA expression in PTC. Immunoblot analysis confirmed the elevated expression levels of five proteins (NPC2, CTSC, GPNMB, DPP4, and ERAP2) in PTC versus benign cystic fluids. Immunohistochemical studies from near 100 pairs of PTC tissue and their adjacent non-tumor counterparts further showed that AGRN (n = 98), CTSC (n = 99), ERAP2 (n = 98) and GPNMB (n = 100) were significantly (p<0.05) overexpressed in PTC and higher expression levels of AGRN and CTSC were also significantly associated with metastasis and poor prognosis of PTC patients. Collectively, our results indicate that an integrated analysis of FNA cystic fluid proteome, cancer cell secretome and tissue transcriptome datasets represents a useful strategy for efficiently discovering novel PTC biomarker candidates.

当前临床常用甲状腺超声与超声引导下细针穿刺活检（ultrasound-guided fine-needle aspiration, USG/FNA）诊断甲状腺乳头状癌（papillary thyroid carcinoma, PTC），但联合其他生物标志物可优化其检测效能。为挖掘新型PTC生物标志物，本研究采用凝胶液相色谱-串联质谱（GeLC-MS/MS）策略，分析了经血清丰度蛋白去除处理的FNA囊液以及2株PTC细胞系的分泌组蛋白质组谱，分别从中鉴定出346、488和2105种蛋白质。比较分析显示，191种蛋白质仅在PTC囊液样本中检出而未在良性囊液样本中出现，因此可能成为潜在的PTC候选生物标志物。在上述蛋白质中，101种可在PTC细胞系分泌组中检出；其中7种蛋白质（NPC2、CTSC、AGRN、GPNMB、DPP4、ERAP2及SH3BGRL3）在公开的PTC转录组数据集中被报道，在4681例PTC样本中呈现上调的mRNA表达。免疫印迹分析证实，相较于良性囊液样本，PTC囊液样本中5种蛋白质（NPC2、CTSC、GPNMB、DPP4及ERAP2）的表达水平显著上调。近100对PTC组织及其配对癌旁非肿瘤组织的免疫组织化学研究进一步显示，AGRN（n=98）、CTSC（n=99）、ERAP2（n=98）及GPNMB（n=100）在PTC组织中均显著过表达（p<0.05）；且AGRN与CTSC的高表达还与PTC患者的肿瘤转移及不良预后显著相关。综上，本研究结果表明，联合分析FNA囊液蛋白质组、癌细胞分泌组及组织转录组数据集，是高效挖掘新型PTC候选生物标志物的可行策略。