Tandem Mass Tag-Based Proteomic Analysis of Normal and Degenerated Human Intervertebral Discs
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ABSTRACT:
SUBMITTER: Fu Y
PROVIDER: S-EPMC10982071 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:PURPOSE: Disc degeneration, and associated low back pain, are a primary cause of disability. Disc degeneration is characterized by dysfunctional cells and loss of proteoglycans: since intervertebral tissue has a limited capacity to regenerate, this process is at present considered irreversible. Recently, cell therapy has been suggested to provide more successful treatment of IVD degeneration. To understand the potential of cells to restore IVD structure/function, tissue samples from degenerated IVD versus healthy discs have been compared. METHODS: Discal tissue from 27 patients (40.17 ± 11 years) undergoing surgery for degenerative disc disease (DDD), DDD + herniation and congenital scoliosis, as controls, was investigated. Cells and matrix in the nucleus pulposus (NP) and annulus fibrosus (AF) were characterized by histology. AF- and NP-derived cells were isolated, expanded and characterized for senescence and gene expression. Three-dimensional NP pellets were cultured and stained for glycosaminoglycan formation. RESULTS: Phenotypical markers of degeneration, such as cell clusters, chondrons, and collagen disorganization were seen in the degenerate samples. In severe degeneration, granulation tissue and peripheral vascularization were observed. No correlation was found between the Pfirrmann clinical score and the extent of degeneration. CONCLUSION: The tissue disorganization in degenerate discs and the paucity of cells out of cluster/chondron association, make the IVD-derived cells an unreliable option for disc regeneration.
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Project description:Esophageal cancer (EC) is a type of extremely aggressive gastrointestinal cancer with high incidences in China and other Asian countries. EC does not have specific symptoms and is relatively easy to metastasize, which makes it difficult in early diagnosis. Thus, novel noninvasive diagnostic method is urgently needed in clinical practice. In this study, mass spectrometry with tandem mass tags and differential protein analysis were applied for identifying esophageal cancer-related proteins. The identified proteins were annotated based on their enrichment in Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, hierarchical clustering was applied based on differentially expressed proteins. As a result, a total of 5131 quantifiable proteins were identified from our liquid chromatography-tandem mass spectrometry with tandem mass tags (LC-MS/MS-TMT) method with 63 upregulated and 97 downregulated differential proteins between esophageal cancer and controlled normal samples. The differentially expressed proteins were highly enriched in GO terms associated with mitochondrial dissemble and apoptosis, and blood vessel regulation, and the upregulated differentially expressed proteins in EC samples were significantly enriched in major histocompatibility complex MHC-class I/II pathway of immune system. The functional clustering analysis revealed potential protein-protein interactions among tetraspanin, myosin, and S-100. In summary, our study provided a practical technological procedure of proteomic analysis for discovering novel biomarkers of a specific cancer type.
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Project description:Intervertebral disc (IVD) degeneration is a leading cause of back pain and precursor to more severe conditions, including disc herniation and spinal stenosis. While traditional growth factor therapies (e.g., TGFβ) are effective at transiently reversing degenerated disc by stimulation of matrix synthesis, it is increasingly accepted that bioscaffolds are required for sustained, complete IVD regeneration. Current scaffolds (e.g., metal/polymer composites, non-mammalian biopolymers) can be improved in one or more IVD regeneration demands: biodegradability, noninvasive injection, recapitulated healthy IVD biomechanics, predictable crosslinking, and matrix repair induction. To meet these demands, tetrazine-norbornene bioorthogonal ligation was combined with gelatin to create an injectable bioorthogonal hydrogel (BIOGEL). The liquid hydrogel precursors remain free-flowing across a wide range of temperatures and crosslink into a robust hydrogel after 5-10 min, allowing a human operator to easily inject the therapeutic constructs into degenerated IVD. Moreover, BIOGEL encapsulation of TGFβ potentiated histological repair (e.g., tissue architecture and matrix synthesis) and functional recovery (e.g., high water retention by promoting the matrix synthesis and reduced pain) in an in vivo rat IVD degeneration/nucleotomy model. This BIOGEL procedure readily integrates into existing nucleotomy procedures, indicating that clinical adoption should proceed with minimal difficulty. Since bioorthogonal crosslinking is essentially non-reactive towards biomolecules, our developed material platform can be extended to other payloads and degenerative injuries.
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Project description:PurposeThe poor prognosis of hepatocellular carcinoma (HCC) urgent us to discover early and effective biomarkers. In this study, we applied tandem mass tag (TMT)-based proteomic analysis to discover potential protein markers for HCC identification and differentiation.Patients and methodsFifteen patients, well-differentiated (G1, N = 5), moderate-differentiated (G2, N = 5), and poorly differentiated (G3, N = 5), with 30 matched pair tissues (both tumor and adjacent non-tumor tissues derived from the same patient) were enrolled. All samples were subjected to TMT labeling and LC-MS/MS analysis. The identified proteins were subsequently assigned to GO and KEGG for predicting function. The identified protein candidates were validated using immunohistochemistry (IHC).ResultsA total of 1010 proteins were identified. Of these, 154 differentially expressed proteins (DEPs), 100 up-regulated and 54 down-regulated, were found between tumor and adjacent non-tumor tissues; 12 DEPs, 9 up-regulated and 3 down-regulated, were found between G1 and G3 tissues; 8 DEPs, 5 up-regulated and 3 down-regulated, were found between G1 and G2 tissues; 11 DEPs, 8 up-regulated and 3 down-regulated, were found between G2 and G3 tissues. Among them, ASS1 and CPS1 were significantly up-regulated while UROD and HBB were significantly down-regulated in G3 compared with G1 and G2 tumors. Three proteins, CYB5A, FKBP11 and YBX1, were significantly up-regulated in G1 compared with both G2 and G3 tumors. The 7 biomarker candidates were further verified by IHC.ConclusionA variety of DEPs related to the histological differentiation of HCC were identified, among which ASS1, CPS1, URPD and HBB proteins were potential biomarkers for distinguishing poorly differentiated HCC, while CYB5A, FKBP11 and YBX1 were potential biomarkers for distinguishing well-differentiated HCC. Our findings may further provide a new insight facilitating the diagnosis and prognosis of HCC.
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Project description:Despite advanced knowledge of the cellular and biomechanical processes of intervertebral disc degeneration (IVDD), the trigger and underlying mechanisms remain unclear. Since the sympathetic nervous system (SNS) has been shown to exhibit catabolic effects in osteoarthritis pathogenesis, it is attractive to speculate that it also influences IVDD. Therefore, we explored the adrenoceptor (AR) expression profile in human IVDs and correlated it with clinical parameters of patients. IVD samples were collected from n = 43 patients undergoing lumbar spinal fusion surgery. AR gene expression was analyzed by semi-quantitative polymerase chain reaction. Clinical parameters as well as radiological Pfirrmann and Modic classification were collected and correlated with AR expression levels. In total human IVD homogenates α1A-, α1B-, α2A-, α2B-, α2C-, β1- and β2-AR genes were expressed. Expression of α1A- (r = 0.439), α2A- (r = 0.346) and β2-AR (r = 0.409) showed a positive and significant correlation with Pfirrmann grade. α1A-AR expression was significantly decreased in IVD tissue of patients with adjacent segment disease (p = 0.041). The results of this study indicate that a relationship between IVDD and AR expression exists. Thus, the SNS and its neurotransmitters might play a role in IVDD pathogenesis. The knowledge of differential AR expression in different etiologies could contribute to the development of new therapeutic approaches for IVDD.
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Project description:Parasitic isopods perforate and attach to the host integument via the mandibles and then feed on hemolymph and exudate from the wounds. Such isopods attack a variety of commercially important fish and crustacean hosts. Similar to other hematophagous parasites, isopods may also employ biomolecules that affect host blood conglutination and defense systems. In the present study, a tandem mass tag-based quantitative proteomic approach was used to identify differentially expressed proteins in Tachaea chinensis parasites of shrimp, by comparing parasitic (fed) and pre-parasitic (unfed) individuals. We identified 888 proteins from a total of 1,510 peptides, with a significant difference in 129 between the fed and unfed groups. Among these, 37 were upregulated and 92 were downregulated in unfed T. chinensis. This indicates that T. chinensis may require more energy before parasitism during its search for a host. In addition, as is the case for other blood-sucking parasites, it might secrete antihemostatic, anti-inflammatory, and immunomodulatory molecules to facilitate blood meal acquisition. To our knowledge, this study is the first to use a TMT-based proteomic approach to analyze the proteome of isopod parasites, and the results will facilitate our understanding of the molecular mechanisms of isopod parasitism on crustaceans.
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Project description:The coupling of the intervertebral disc (IVD) and vertebra as a biomechanical unit suggests that changes in the distribution of pressure within the IVD (intradiscal pressure, IDP) as a result of disc degeneration can influence the distribution of bone density within the vertebra, and vice versa. The goal of this study was to assess the correspondence between IDP and bone density in the adjacent vertebrae, with emphasis on how this correspondence differs between healthy and degenerated IVDs. Bone density of the endplates and subchondral bone in regions adjacent to the anterior and posterior annulus fibrosus (aAF and pAF, respectively) and nucleus pulposus (NP) was measured via quantitative computed tomography (QCT) in 61 spine segments (T7-9, T9-11, T10-12; 71±14years). IDP was measured in the aAF, NP, and pAF regions in 26 of the spine segments (68±16years) while they were tested in flexed (5°) or erect postures. Disc degeneration was assessed by multiple grading schemes. No correlation was found between bone density and IDP in either posture (p>0.104). Regional variations in IDP and, to a greater extent bone density, were found to change with advancing degeneration: both IDP (p=0.045) and bone density (p=0.024) decreased in the NP region relative to the aAF region. The finding of only a modest correspondence between degeneration-associated changes in IDP and bone density may arise from complexity in how IDP relates to mechanical force transmission through the endplate and from limitations of the available IVD grading schemes in estimating the mechanical behavior of the IVD.
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Project description:Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics─currently on the order of 25 mg wet tissue weight. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is ≥10 million peripheral blood mononuclear cells (PBMCs). Across large study cohorts, this requirement will exceed what is obtainable for many individual patients/time points. For this reason, we were interested in the impact of differential peptide loading across multiplex channels on proteomic data quality. To achieve this, we tested a range of channel loading amounts (approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification precision, and peptide/phosphopeptide detection in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, fewer missing values were observed in TMT channels with higher peptide loading amounts compared to lower loadings. Moreover, channels with a lower loading have greater quantitative variability than channels with higher loadings. A statistical analysis showed that decreased loading amounts result in an increase in the type I error rate. We then examined the impact of differential loading on the detection of known differences between distinct AML cell lines. Similar patterns of increased data missingness and higher quantitative variability were observed as loading was decreased resulting in fewer statistical differences; however, we found good agreement in features identified as differential, demonstrating the value of this approach.
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Project description:BackgroundCryptosporidium andersoni initiates infection by releasing sporozoites from oocysts through excystation. However, the proteins involved in excystation are unknown. Determining the proteins that participate in the excystation of C. andersoni oocysts will increase our understanding of the excystation process.MethodsCryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis, were used to investigate the proteomic expression profiles of C. andersoni oocysts before and after excystation.ResultsProteomic analysis identified a total of 1586 proteins, of which 17 were differentially expressed proteins (DEPs) upon excystation. These included 10 upregulated and seven downregulated proteins. The 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time RT-PCR of eight selected genes validated the proteomic data.ConclusionsThis study provides information on the protein composition of C. andersoni oocysts as well as possible excystation factors. The data may be useful in identifying genes for diagnosis, vaccine development, and immunotherapy for Cryptosporidium.
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Project description:Pediatric obstructive sleep apnea (OSA) is a frequent respiratory disorder with an estimated prevalence of 3-6% in the general population. However, the underlying pathophysiology of OSA remains unclear. Recently, proteomic analysis using high-resolution and high-throughput mass spectrometry has been widely used in the field of medical sciences. In the present study, tandem mass tag (TMT)-based proteomic analysis was performed in the serum of patients with OSA. The proteomic analysis revealed a set of differentially expressed proteins that may be associated with the pathophysiology of OSA. The differentially expressed proteins in patients with OSA were enriched in pathways including phagosome and glycan synthesis/degradation, immune response, and the hedgehog signaling pathway, indicating that such functions are key targets of OSA. Moreover, the experimental validation studies revealed that four proteins including ANTXR1, COLEC10, NCAM1, and VNN1 were reduced in the serum from patients with moderate and severe OSA, while MAN1A1 and CSPG4 protein levels were elevated in the serum from patients with severe OSA. The protein levels of ANTXR1, COLEC10, NCAM1, and VNN1 were inversely correlated with apnea-hypopnea index (AHI) in the recruited subjects, while the protein level of MAN1A1 was positively correlated with AHI, and no significant correlation was detected between CSPG4 protein and AHI. In summary, the present study for the first time identified differentially expressed proteins in the serum from OSA patients with different severities by using TMT-based proteomic analysis. The functional enrichment studies suggested that several signaling pathways may be associated with the pathophysiology of OSA. The experimental validation results indicated that six proteins including ANTXR1, COLEC10, NCAM1, VNN1, CGPG4, and MAN1A1 may play important roles in the pathophysiology of OSA, which requires further mechanistic investigation.
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