Characterization of the seminal plasma proteome in men with prostatitis by mass spectrometry

Background  

Prostatitis is an inflammation of the prostate gland which affects approximately 10% of men. Despite its frequency, diagnosing prostatitis and monitoring patient response to treatment remains frustrating. As the prostate contributes a substantial percentage of proteins to seminal plasma, we hypothesized that a protein biomarker of prostatitis might be found by comparing the seminal plasma proteome of patients with and without prostatitis.     

Mass spectrometry is only one piece of the puzzle in clinical proteomics.

Biomarker discovery in clinical proteomics is being performed on relatively large patient cohorts by utilizing the high throughput of laser desorption/ionization mass spectrometry (MALDI- and SELDI-TOF-MS). Dealing directly with patient samples as opposed to working in cell or animal systems requires a host of considerations both before and after mass spectrometric analysis to obtain robust biomarker candidates. The challenges associated with the heterogeneity of typical samples are amplified by the ability to detect hundreds to thousands of proteins simultaneously. Adherence to protocols and consistency, however, can ensure optimal results. A study starts necessarily with a relevant clinical question and proceeds to a planning phase where sample availability, statistical test selection, logistics and bias reduction are key points. The physical analysis requires consistency and standardized protocols that are helped significantly through automation. Data analysis is broken into two stages, screening and final testing, which can detect either single candidates or a pattern of proteins. Biomarker identification can be performed at this point and will help significantly in the last stage, interpretation. Replication should be performed in an independent sample set in a separate study. The candidate biomarkers from an initial study give a wealth of information that can help to pinpoint patient subpopulations for a more exhaustive proteomic study using complementary platforms with limited capacity but extremely high information content. A clinical proteomics pilot project can also lead to better selection of model systems by providing a direct link with patient samples.     

Development of high throughput dispersive LC-ion mobility-TOFMS techniques for analysing the human plasma proteome.

A technique that combines ion mobility spectrometry (IMS) with reversed-phase liquid chromatography (LC), collision-induced dissociation (CID) and mass spectrometry (MS) has been developed. The approach is described as a high throughput means of analysing complex mixtures of peptides that arise from enzymatic digestion of protein mixtures. In this approach, peptides are separated by LC and, as they elute from the column, they are introduced into the gas phase and ionised by electrospray ionisation. The beam of ions is accumulated in an ion trap and then the concentrated ion packet is injected into a drift tube where the ions are separated again in the gas phase by IMS, a technique that differentiates ions based on their mobilities through a buffer gas. As ions exit the drift tube, they can be subjected to collisional activation to produce fragments prior to being introduced into a mass spectrometer for detection. The IMS separation can be carried out in only a few milliseconds and offers a number of advantages compared with LC-MS alone. An example of a single 21-minute LC-IMS-(CID)-MS analysis of the human plasma proteome reveals approximately 20,000 parent ions and approximately 600,000 fragment ions and evidence for 227 unique protein assignments.     

The synaptic vesicle proteome

Synaptic vesicles are key organelles in neurotransmission. Vesicle integral or membrane-associated proteins mediate the various functions the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the pre-synaptic plasma membrane during exo- and endocytosis. Within the past two decades, converging work from several laboratories resulted in the molecular and functional characterization of the proteinaceous inventory of the synaptic vesicle compartment. However, up until recently and due to technical difficulties, it was impossible to screen the entire organelle thoroughly. Recent advances in membrane protein identification and mass spectrometry (MS) have dramatically promoted this field. A comparison of different techniques for elucidating the proteinaceous composition of synaptic vesicles revealed numerous overlaps but also remarkable differences in the protein constituents of the synaptic vesicle compartment, indicating that several protein separation techniques in combination with differing MS approaches are required to identify and characterize the synaptic vesicle proteome. This review highlights the power of various gel separation techniques and MS analyses for the characterization of the proteome of highly purified synaptic vesicles. Furthermore, the newly detected protein assignments to synaptic vesicles, especially those proteins which are new to the inventory of the synaptic vesicle proteome, are critically discussed.     

Proteomic analysis suggests that monoterpenes in lemongrass disrupt Ca2+ homeostasis in Haemaphysalis longicornis leading to mitochondrial depolarization and cytotoxicity

Complex mixtures of bioactive ingredients in plant essential oils present complex chemistries which involve different modes of action. An increasing body of scientific reports has recently focused on the acaricidal activities of plant essential oils attributed to their monoterpene components, but information about their underlying molecular mechanism of action is scarce. Here, after the chemical analysis of lemongrass oil, a proteomic analysis of the ovary, salivary gland, and midgut of Haemaphysalis longicornis exposed to Cymbopogon citratus (lemongrass) essential oil was performed via data-independent acquisition mass spectrometry (DIA-MS) technology to further elucidate the molecular mechanisms involved. Pathway analysis reveals the activation of metabolic pathways mediated by oxidoreductases and transferases. Furthermore, the upregulation of various calcium-associated proteins and the upregulation of cytochrome c1, cytochrome c oxidase polypeptide IV, and programmed cell death protein 6-like isoform X1 suggest a cytotoxic mode of action via the formation of reactive oxygen species (ROS), mitochondrial Ca²⁺ overload, mitochondrial uncoupling, and depolarization, and ATP depletion leading to either apoptotic or necrotic death. Morphological alterations observed after the RNAi of a major detoxification enzyme (glutathione S-transferase) merit further investigation. Hence, the cytotoxic mode of action exhibited by C. citratus oil could be vital for the development of eco-friendly acaricide.     

The third Central and Eastern European Proteomic conference

The third Central and Eastern European Proteomic Conference was held at Hotel Benzcur, Budapest, Hungary, from the 6–9 October 2009. The meeting was the third in a series of proteomic conferences to be held in this region of Europe, with the key aim of strengthening the links with scientists from Central and Eastern Europe, as well as international groups worldwide. It was attended by more than 150 delegates from various countries and many proteomic topics, including biomarker discovery, post-translational modifications, clinical proteomics, as well as new proteomic technologies, which may facilitate future progress, were discussed over the 3 days.     

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

Understanding tenderness variability and ageing changes in buffalo meat: biochemical,ultrastructural and proteome characterization

Understanding of biological impact of proteome profile on meat quality is vital for developing different approaches to improve meat quality. Present study was conducted to unravel the differences in biochemical, ultrastructural and proteome profile of longissimus dorsi muscle between buffaloes (Bubalus bubalis) of different age groups (young v. old). Higher (P<0.05) myofibrillar and total protein extractability, muscle fibre diameter, and Warner-Bratzler shear force (WBSF) values was observed in old buffalo meat relative to meat from young buffaloes. Scanning electron microscopy photographs revealed reduced fibre size with increased inter-myofibrillar space in young compared with old buffalo meat. Transmission electron microscopy results revealed longer sarcomeres in young buffalo meat relative to meat from old buffaloes. Proteomic characterization using two-dimensional gel electrophoresis (2DE) found 93 differentially expressed proteins between old and young buffalo meat. Proteome analysis using 2DE revealed 191 and 95 differentially expressed protein spots after 6 days of ageing in young and old buffalo meat, respectively. The matrix assisted laser desorption ionization time-of flight/time-of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of selected gel spots helped in identifying molecular markers of tenderness mainly consisting of structural proteins. Protein biomarkers identified in the present study have the potential to differentiate meat from young and old buffaloes and pave the way for optimizing strategies for improved buffalo meat quality.     

The human urinary proteome: combinational approaches to comprehensive mapping

The development methodologies for the assessment of the protein content of biological samples have been in the ‘eye of the storm’ in proteomics for almost two decades. The work of Zerefos et al. is a continuation of this trend, focusing on analysis of urinary proteins using a combination of separation methodologies. In this work, the authors employ a previously analyzed control urine sample. Three different methodologies are presented, involving the combination of classical separation approaches, such as SDS-PAGE, preparative electrophoresis and liquid chromatography and standard mass spectrometer instrumentation. Several hundred proteins were reported following the application of a typical proteomics workflow and the use of a data meta-analysis platform to enhance the credibility of the final output. This is also established through cross-method (within this study) as well as cross-study (comparison of this with other main studies in the field) data comparisons. Emphasis is placed on the presentation of experimental identifiers as well as information provided at the peptide level.     

脑脊液蛋白质组技术及临床应用研究进展

脑脊液(CSF)围绕并支持中枢神经系统(CNS),包括脑室和蛛网膜下腔,由于脑脊液与中枢神经系统直接接触,所以其是寻找中枢神经系统疾病生物标记物的重要来源。国内外学者开展了大量CSF蛋白质组学的研究工作,并取得了较大进展。文中综述了近年来CSF蛋白质组学技术及临床应用研究进展。     

The oxidized thiol proteome in aging and cataractous mouse and human lens revealed by ICAT labeling

Age‐related cataractogenesis is associated with disulfide‐linked high molecular weight (HMW) crystallin aggregates. We recently found that the lens crystallin disulfidome was evolutionarily conserved in human and glutathione‐depleted mouse (LEGSKO) cataracts and that it could be mimicked by oxidation in vitro (Mol. Cell Proteomics, 14, 3211‐23 (2015)). To obtain a comprehensive blueprint of the oxidized key regulatory and cytoskeletal proteins underlying cataractogenesis, we have now used the same approach to determine, in the same specimens, all the disulfide‐forming noncrystallin proteins identified by ICAT proteomics. Seventy‐four, 50, and 54 disulfide‐forming proteins were identified in the human and mouse cataracts and the in vitro oxidation model, respectively, of which 17 were common to all three groups. Enzymes with oxidized cysteine at critical sites include GAPDH (hGAPDH, Cys247), glutathione synthase (hGSS, Cys294), aldehyde dehydrogenase (hALDH1A1, Cys126 and Cys186), sorbitol dehydrogenase (hSORD, Cys140, Cys165, and Cys179), and PARK7 (hPARK7, Cys46 and Cys53). Extensive oxidation was also present in lens‐specific intermediate filament proteins, such as BFSP1 and BFSP12 (hBFSP1 and hBFSP12, Cys167, Cys65, and Cys326), vimentin (mVim, Cys328), and cytokeratins, as well as microfilament and microtubule filament proteins, such as tubulin and actins. While the biological impact of these modifications for lens physiology remains to be determined, many of these oxidation sites have already been associated with either impaired metabolism or cytoskeletal architecture, strongly suggesting that they have a pathogenic role in cataractogenesis. By extrapolation, these findings may be of broader significance for age‐ and disease‐related dysfunctions associated with oxidant stress.     

基于蛋白质组学技术的新型冠状病毒肺炎研究进展

新型冠状病毒肺炎（coronavirus disease 2019,COVID-19）是一种由严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引发的传染病。此种病毒传染性强、传播速度快,对全球人民的身体健康和生命安全造成严重威胁。蛋白质组学技术以其高通量、高灵敏度的特点,在疾病生物标志物的发现、分子机制研究及治疗靶点研究中扮演着重要角色,并被广泛应用于COVID-19的研究中。本文介绍了SARS-CoV-2的基因组结构及病毒感染过程,总结了目前常用的基于质谱的蛋白质组学研究技术,重点综述了蛋白质组学技术在COVID-19生物标志物的发现、分子机制研究和药物治疗靶标研究中的应用进展,最后展望了蛋白质组学的未来发展方向,以期能够有助于推动蛋白质组学技术在COVID-19精准诊断和治疗中的发展。     

真核细胞质膜蛋白质组研究进展

细胞膜（质膜）蛋白质是细胞的“门铃”与“门户”,是许多药物的作用靶标。细胞质膜蛋白质组的研究正成为蛋白质组研究的热点,这方面的研究有利于具有重要功能的低丰度蛋白质的发掘,为药物研发和疾病的诊断提供靶体与标记蛋白质。然而,质膜蛋白质组的研究在强疏水性跨膜蛋白质和低丰度膜蛋白质的分离和鉴定上遇到了方法学的挑战。本文对质膜及其微区的纯化、质膜蛋白质组的分离与鉴定、生物信息学,以及亚细胞定位研究的近期进展作扼要介绍。     

生物质谱与蛋白质组学

蛋白质组学是后基因组学时代最受关注的研究领域之一,其核心的鉴定技术——生物质谱近年来在仪器设计以及鉴定通量、分辨率和灵敏度等各方面均有质的飞跃,促进了蛋白质表达谱作图、定量蛋白质组分析、亚细胞器蛋白质组作图、蛋白质翻译后修饰以及蛋白质相互作用等蛋白质组研究各个领域的飞速发展。本综述了生物质谱技术的最新进展,及其在蛋白质组学研究中的应用。     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.