Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given “protein” is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (Mr). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and Mr. Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes.
The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of complications and requires adjuvant radiotherapy and medications. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. The study aimed to proteomic variations of invasive and non-invasive NFPAs and sought the protein markers for invasive NFPAs. Invasive (n = 4) and non-invasive (n = 4) NFPA tissues were analyzed (n = 3-5/each tissue) with 2DE and PDQuest software. Twenty-four high-resolution 2DE gels were quantitatively compared to determine differentially expressed proteins (DEPs) between invasive and non-invasive NFPAs. Approximately 1200 protein spots were detected in each 2DE map, and 103 differential spots (64 upregulated and 39 downregulated) were identified. Among those 103 differential spots, 57 DEPs (30 upregulated and 27 downregulated) were characterized with peptide mass fingerprint and MS/MS. Gene-ontology (GO) and ingenuity pathway analyses of those DEPs revealed pathway networks including mitochondrial dysfunction, oxidative stress, mitogen-activated protein kinase signaling abnormality, TR/RXR activation, proteolysis abnormality, ketogenesis and ketolysis, cyclin-dependent kinase C signaling abnormality, and amyloid processing that were significantly associated with invasive characteristics of invasive NFPA. Those data demonstrate that proteomic variations exist between invasive and non-invasive NFPAs. 2DE-based comparative proteomics is an effective approach to identify proteomic variations and pathway network variations. Those findings will serve as a basis to understand the molecular mechanisms of invasive NFPAs and to discover protein markers to effectively manage patients with invasive NFPAs.
Non-functional pituitary adenoma (NFPA) seriously affects hypothanamus-pituitary-target organ axis system, with a series of molecule alterations in the multiple levels of genome, transcriptome, proteome, and post-translational modifications, and those molecules mutually interact in a molecular-network system. Meta analysis coupled with IPA pathway-network program was used to comprehensively analyze nine sets of documented NFPA omics data, including NFPA quantitative transcriptomics data [280 differentially expressed genes (DEGs)], NFPA quantitative proteomics data [50 differentially expressed proteins (DEPs)], NFPA mapping protein data (218 proteins), NFPA mapping protein nitration data (9 nitroproteins and 3 non-nitrated proteins), invasive NFPA quantitative transriptomics data (346 DEGs), invasive NFPA quantitative proteomics data (57 DEPs), control mapping protein data (1469 proteins), control mapping protein nitration data (8 nitroproteins), and control mapping phosphorylation data (28 phosphoproteins). A total of 62 molecular-networks with 861 hub-molecules and 519 canonical-pathways including 54 cancer-related canonical pathways were revealed. A total of 42 hub-molecule panels and 9 canonical-pathway panels were identified to significantly associate with tumorigenesis. Four important molecular-network systems, including PI3K/AKT, mTOR, Wnt, and ERK/MAPK pathway-systems, were confirmed in NFPAs by PTMScan experiments with altered expression-patterns and phosphorylations. Nineteen high-frequency hub-molecules were also validated in NFPAs with PTMScan experiment with at least 2.5-fold changes in expression or phosphorylation, including ERK, ERK1/2, Jnk, MAPK, Mek, p38 MAPK, AKT, PI3K complex, p85, PKC, FAK, Rac, Shc, HSP90, NFκB Complex, histone H3, AP1, calmodulin, and PLC. Furthermore, mTOR and Wnt pathway-systems were confirmed in NFPAs by immunoaffinity Western blot analysis, with significantly decreased expression of PRAS40 and increased phosphorylation levels of p-PRAS40 (Thr246) in mTOR pathway in NFPAs compared to controls, and with the decreased protein expressions of GSK-3β and GSK-3β, significantly increased phosphorylation levels of p-GSK3α (Ser21) and p-GSK3β (Ser9), and increased expression level of β-catenin in Wnt pathway in NFPAs compared to Long et al. Molecular Networks of Non-functional Pituitary Adenomas controls. Those findings provided a comphrensive and large-scale pathway network data for NFPAs, and offer the scientific evidence for insights into the accurate molecular mechanisms of NFPA and discovery of the effective biomarkers for diagnosis, prognosis, and determination of therapeutic targets.
Protein ubiquitination is an important post-translational modification that is associated with multiple diseases, including pituitary adenomas (PAs). Protein ubiquitination profiling in human pituitary and PAs remains unknown. Here, we performed the first ubiquitination analysis with an anti-ubiquitin antibody (specific to K-ε-GG)-based label-free quantitative proteomics method and bioinformatics to investigate protein ubiquitination profiling between PA and control tissues. A total of 158 ubiquitinated sites and 142 ubiquitinated peptides in 108 proteins were identified, and five ubiquitination motifs were found. KEGG pathway network analysis of 108 ubiquitinated proteins identified four statistically significant signaling pathways, including PI3K-AKT signaling pathway, hippo signaling pathway, ribosome, and nucleotide excision repair. R software Gene Ontology (GO) analysis of 108 ubiquitinated proteins revealed that protein ubiquitination was involved in multiple biological processes, cellular components, and molecule functions. The randomly selected ubiquitinated 14-3-3 zeta/delta protein was further analyzed with Western blot, and it was found that upregulated 14-3-3 zeta/delta protein in nonfunctional PAs might be derived from the significantly decreased level of its ubiquitination compared to control pituitaries, which indicated a contribution of 14-3-3 zeta/delta protein to pituitary tumorigenesis. These findings provided the first ubiquitinated proteomic profiling and ubiquitination-involved signaling pathway networks in human PAs. This study offers new scientific evidence and basic data to elucidate the biological functions of ubiquitination in PAs, insights into its novel molecular mechanisms of pituitary tumorigenesis, and discovery of novel biomarkers and therapeutic targets for effective treatment of PAs.
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