Proteomics
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Mammalian cells secrete two types of extracellular vesicles either constitutively or in a regulated manner: exosomes (50-100 nm in diameter) released from the intracellular compartment and ectosomes (also called microvesicles, 100-1000 nm in diameter) shed directly from the plasma membrane. Extracellular vesicles are bilayered proteolipids enriched with proteins, mRNAs, microRNAs, and lipids. In recent years, much data have been collected regarding the specific components of extracellular vesicles from various cell types and body fluids using proteomic, transcriptomic, and lipidomic methods. ⋯ These results provide valuable information on the molecular mechanisms involved in vesicular cargo-sorting and biogenesis. Furthermore, studies of these complex extracellular organelles have facilitated conceptual advancements in the field of intercellular communication under physiological and pathological conditions as well as for disease-specific biomarker discovery. This review focuses on the proteomic, transcriptomic, and lipidomic profiles of extracellular vesicles, and will briefly summarize recent advances in the biology, function, and diagnostic potential of vesicle-specific components.
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Exosomes and microvesicles (MVs) are nanometer-sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro-environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro-RNAs; oncogenic cargo hence often referred to as oncosomes). ⋯ This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV-associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio-fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.
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Each phase of eukaryotic cell cycle is tightly controlled by multicomponent regulatory networks based on complex relationships of protein phosphorylation. In order to better understand the relationships between kinases and their substrate proteins during the progression of cell cycle, we analyzed phosphoproteome of HeLa cells during G1, S, and G2/M phases of cell cycle using our developed quantitative phosphoproteomic approaches. A total of 4776 high-confidence phosphorylation sites (phosphosites) in 1177 proteins were identified. ⋯ Further analyses showed that approximately half of the examined two phosphosite combinations were correlatively regulated, regardless of whether the kinase groups were same or not. In general, the majority of proteins containing correlated phosphosites had solely co-regulated or counter-regulated phosphosites, and co-regulation was significantly more frequent than counter-regulation, suggesting that the former may be more important for regulating the cell cycle. In conclusion, our findings provide new insights into the complex regulatory mechanisms of protein phosphorylation networks during eukaryotic cell cycle.
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Clinical Trial
Proteomic analysis of cerebrospinal fluid before and after intrathecal injection of steroid into patients with postherpetic pain.
Postherpetic neuralgia (PHN) is the most frequent complication of herpes zoster, and the risk of it increases with age. By comparing proteomes of the cerebrospinal fluid (CSF) before and after the treatment, it may be possible to identify proteins that play a role in PHN and to predict responses to various treatments. To address this issue, we enrolled eight outpatients with PHN over 55 years of age and treated them with intrathecal methylprednisolone and lidocaine four times every week, collecting CSF samples before the treatment at each visit. ⋯ Of 145 differentially expressed spots, the levels of nine proteins were decreased by the treatment including lipocalin-type prostaglandin D synthase (L-PGDS), and five were increased by it. The time course of alterations in the L-PGDS concentration in the CSF of each patient, detected by a pairwise and sandwich ELISA by SPR constructed here was well correlated with that by 1DE Western blots with anti-L-PGDS antibody, but was not related with that of the pain relief. The present study demonstrates that the real-time ELISA was precise and sensitive enough to measure L-PGDS in the CSF and that the steroid treatment decreased the L-PGDS concentration in CSF.
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We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. ⋯ In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.