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Altered profile of glycosylated proteins in serum samples obtained from patients with Hashimoto′s thyroiditis following depletion of highly abundant proteins

ABSTRACT: Objectives: Hashimoto’s thyroiditis (HT) is one of the most common autoimmune disorders; however, its underlying pathological mechanisms remain unclear. Although aberrant glycosylation has been implicated in the N-glycome of immunoglobulin G (IgG), changes in serum proteins have not been comprehensively characterized. This study aimed to investigate the glycosylation profile of serum samples of HT patients that were depleted of highly abundant proteins by and propose the potential functions of glycoproteins for the further study of pathological mechanisms of HT . Methods: A lectin microarray containing 70 lectins was used to detect and analyze glycosylation of serum proteins using serum samples (N=27 HT; N=26 healthy control [HC]) depleted of abundant proteins. Significant differences in glycosylation status between HT patients and the HC group were verified by lectin blot analysis. A lectin-based pull-down assay combined with mass spectrometry was used to investigate potential glycoproteins combined with differentially present lectins, and an enzyme-linked immunosorbent assay (ELISA) was used to identify the expression of targeted glycoproteins in 131 patients with papillary thyroid carcinoma (PTC), 131 patients with benign thyroid nodules (BTN) patients, 130 patients with HT, and 128 HCs. Results: Compared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the Vicia villosa agglutinin (VVA) binding signal was increased. The difference in VVA binding signals verified by lectin blotting was consistent with the results of the lectin microarray. A total of 113 potential VVA-binding glycoproteins were identified by mass spectrometry and classified by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses . Using ELISA, we confirmed that lactoferrin (LTF) and mannan-binding lectin-associated serine protease 1 (MASP-1) levels were elevated in the serum of patients with HT and PTC. Conclusions: Following depletion of abundant proteins, remaining serum proteins in HT patients exhibited lower glycosylation levels than those observed in HCs. An increased level of potential VVA-binding glycoproteins may play an important role in HT development. LTF and MASP-1 expression was significantly higher in the serum of HT and PTC patients, suggesting key roles played by glycosylation in pathological mechanisms underlying HT and PTC.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

SUBMITTER: Yaozheng Xu

LAB HEAD: Xiaosong Qin

PROVIDER: PXD040847 | Pride | 2023-06-22

REPOSITORIES: Pride

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Project description:PURPOSE: Amino acids (AAs) play important physiological roles in living cells. Some amino acid changes in blood are specific for autoimmune disorders, and some are specific for thyroid cancer. The aims of this study were to profile AA metabolites in the serum of patients with papillary thyroid carcinoma (PTC0) without Hashimoto’s thyroiditis (HT) and patients with PTC with HT (PTC1) and predict whether AA metabolites are associated with thyroid disease, thyroid hormone and thyroid autoantibodies. METHODS: A total of 93 serum samples were collected, including 28 healthy controls (HCs), 28 PTC0 patients and 37 PTC1 patients. Serum samples were analysed by high-performance liquid chromatography-triple stage quadrupole-mass spectrometry (HPLC-TSQ-MS), and 21 amino acids (AAs) were detected.RESULTS: The serum concentration of glutamic acid was significantly elevated in PTC1 patients compared with PTC0 patients. Lysine was the second amino acid that differentiated these two groups of PTC patients. In addition, the serum concentrations of glycine, alanine and tyrosine were significantly reduced in both PTC patient groups compared to the HC group. These AAs were also correlated with thyroid hormones and antibodies. Five amino acid markers, namely, glycine, tyrosine, glutamic acid, glutamine and arginine, separated/distinguished PTC0 patients from healthy subjects, and eight AA markers, the same AAs as above without arginine but with alanine, leucine, valine and histidine, separated/distinguished PTC1 patients from healthy subjects based on ROC analysis.CONCLUSION: Compared with the HCs, changes in AAs in PTC0 and PTC1 patients showed similar patterns, suggesting the possibility of a common pathophysiological basis, which confirms preliminary research that PTC is significantly associated with pathologically confirmed HT. We found two AAs, lysine and alanine, that can perform diagnostic functions in distinguishing PTC1 from PTC0.

Project description:Glycosylation of proteins is one of the most common post-translational modification (PTM) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of the tear film might promote development of chronic eye diseases indicating glycoproteins as valuable source for biomarker discovery or drug target identification. The present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and the characterization of their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we accumulated first native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA and UEA I, termed as 4L) was applied for glycopeptide enrichment from human tear sample digests followed by MS-based detection and localization of their specific N-glycosylation sites. As main result, the present study identified in total 26 N glycosylation sites of 11 N-glycoproteins in tear sample pools of healthy individuals (n=3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP) as well as extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, which were already associated with the pathogenesis of various chronic eye diseases such as the dry eye syndrome (DES). In conclusion, the results of the present study will serve as important tear film N-glycoprotein catalogue for future studies focusing the human tear film and ocular surface-related inflammatory diseases.

Project description:Context: Papillary thyroid cancer (PTC) is the most common thyroid cancer with dramatically increasing worldwide. Accurate diagnosis is crucial to avoid unnecessary overtreatment for low-risk patients, but the currently available markers are still insufficient. tRNA-derived fragments (tRFs) are emerging as a new class of small non-coding RNAs due to their potential roles as novel biomarkers and therapeutic targets in cancer. However, their involvement in papillary thyroid carcinoma (PTC) is still unclear. Objectives: This study was performed to identify differentially expressed tRFs in PTC and investigate the role of serum tRFs to discriminate PTC from nodular goiters (NGs).Methods and Materials: tRF expression profiles were measured in pooled sera from patients with PTCs (n=6), NGs (n=6) and healthy subjects (n=6) using high-throughput sequencing (cohort1). One selected tRF candidate was validated in the same individual samples (cohort1) by qRT-PCR. The confirmed tRF was further validated in a larger second cohort including patients with PTCs (n=30), NGs (n=20) and healthy individuals (n=18).Results: Our sequencing results showed that circulating tRFs were differentially expressed in patients with PTC compared with those with NG and healthy people. After the validation in individual samples, tRF-Pro-AGG-018 was confirmed as differentially elevated in serum samples of patients with PTC. These results were further confirmed in a larger second cohort. Moreover, tRF-Pro-AGG-018 exhibited good diagnostic performance with a sensitivity of 0.7222 and a specificity of 0.8846 for PTC and it was associated with several targeted genes of PTC. Conclusions: Our study identified that the serum tRFs were differentially expressed in PTC compared with NG and healthy individuals. Notably, circulating tRF-Pro-AGG-018 was found to have a significant elevation in patients with PTC and exhibited good diagnostic performance with relatively high sensitivity and specificity for PTC. Our findings suggest tRFs might serve as novel circulating biomarkers in predicting patients with PTC, which may prevent unnecessary thyroid surgeries for low-risk and benefit from optimal

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Altered profile of glycosylated proteins in serum samples obtained from patients with Hashimoto′s thyroiditis following depletion of highly abundant proteins

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