蛋白质组学在消化系疾病中医药研究中的应用

自然界各种生命物质都是由蛋白质组成的,各种蛋白质按不同的组成、结构和修饰,组成各种细胞和生命物质.蛋白质组学通过从整体角度分析细胞内动态变化的蛋白质组成、表达水平、修饰状况、蛋白质之间以及蛋白质与其他生物分子之间的相互作用,从而了解蛋白质的功能及其在生命过程中的作用.它包括双向凝胶电泳、生物质谱鉴定、生物信息学.当人体中某部分发生生理、病理变化时,该机体组织的蛋白质组发生变化,或代谢物的蛋白质组发生变化,就可以通过蛋白质组学技术找到这些新产生的蛋白质(称为"上调"或"表达增高)或减少甚至消失的蛋白质(称为"下调"或"表达降低"),这些蛋白质叫做"差异蛋白",是用于研究生理、病理变化诊断的潜在"靶点".     

蛋白质组学研究方法

随着人类功能基因组计划的实施和推进，以蛋白质组(Proteome)和蛋白质组学(Proteomics)的提出为标志，人类从蛋白质水平全面揭示生命本质的研究进入一个全新的领域。“工欲善其事，必先利其器”，近年来蛋白质组学的迅猛发展与研究方法的改进和研究手段的提高是密不可分的。蛋白质组学的发展是伴随着蛋白质研究技术，尤其是双向凝胶电泳(2-D凝胶电泳)和新型质谱技术以及生物信息学的发展而发展的。本文将对主要蛋白质组学技术作一概述。     

蛋白质组学在消化系肿瘤中的应用

蛋白质组学是以双向凝胶电泳和质谱技术为核心, 从整体的角度研究生物机体、组织、细胞甚至细胞器基因编码的全部蛋白质, 在更贴近生命本质的层次上发现和理解生命活动的规律.作为肿瘤研究的新平台, 蛋白质组学对食管癌、胃癌、肝癌、胰腺癌、结直肠癌等消化系肿瘤的早期诊断, 寻找新标志物, 治疗以及药物开发的新靶点等方面有重要意义, 具有潜在、广阔的应用前景.     

蛋白质组学技术在风湿性疾病研究中的应用

蛋白质组学是在蛋白质水平定量、动态、整体的研究生物体的一门新兴学科。双向电泳技术、质谱技术和生物信息学是蛋白质组学的三大支撑技术。蛋白质组学技术已应用于类风湿关节炎、干燥综合征等多种风湿性疾病的研究,旨在发现早期诊断标志物,评价治疗效果及判断预后。     

蛋白质组学在食管癌发生中的研究进展

随着人类基因组全序列草图的完成,从基因水平向蛋白质水平的深化,已成为生命科学研究的迫切需要和新的任务。蛋白质组学是研究蛋白质组成和动态变化的一门新兴学科,蛋白质组学研究技术已经应用于肿瘤的发生机制、诊断、治疗之中。从蛋白质整体水平上研究食管癌的发生与转移,寻找食管癌发生及转移相关的新的蛋白质、特异性的标志物及药物治疗的靶标,对食管癌的诊治将起到重要作用。现综述蛋白质组学研究在食管癌发生中的研究进展。     

SELDI-TOF-MS技术在消化道肿瘤诊断中的应用

蛋白质组学（proteomics）是一门以全部蛋白质性质研究为基础，从蛋白质水平进一步认识生命活动的机理和疾病发生的机制，蛋白质组研究是对生物体在蛋白质水平定量、动态、整体性的研究，是继基因组学后一个重要的研究热点。随着蛋白组学技术、高同量技术及生物信息技术的飞速发展，蛋白质芯片表面加强激光解吸电离．飞行时间．质谱 （ surface enhanced laser desorption/ionization-time of flight-mass spectrometry, SELDI-TOF-MS） 技术应运而生。它是蛋白质组学中用于筛选和鉴定一种蛋白质标志物的新技术，目前已在某些肿瘤生物标志物筛选方面取得突破性进展，并有效地应用于癌症的早期诊断.     

蛋白质组学在人类疾病研究中的应用

蛋白质组学是后基因组时代生命科学研究的热点和前沿领域,应用蛋白质组学对临床疾病进行研究,不仅从蛋白质水平上揭示疾病的本质,还有助于全面探讨其病理生理机制,寻找诊断和预后标志物,发现药物治疗靶点。目前蛋白质组学广泛用于肿瘤、心血管疾病和肾脏疾病等领域。     

蛋白质组学在胰腺疾病研究中的应用

蛋白质组学是研究蛋白质的表达、翻译后修饰、在细胞内定位以及蛋白质与蛋白质之间的相互作用的科学。传统的研究手段主要采用双向凝胶电泳和质谱技术,前者用于蛋白质的分离,后者用于蛋白质的鉴定。目前新兴的研究技术主要有相差凝胶电泳技术同位素亲和标签技术。蛋白质组学在胰腺疾病的研究主要是通过正常个体与患病个体的血清、胰液或组织等进行蛋白质比较,发现差异蛋白质,从而对急、慢性胰腺炎和胰腺癌等胰腺疾病的发病机制、早期诊断和有效治疗提供理论依据和新思路。     

MALDI-TOF-MS技术应用于肝细胞癌蛋白质组学的研究进展

蛋白质组学（proteomics）是从整体水平对蛋白质进行研究的一门重要学科。蛋白质组学技术为开展HCC研究建立了新的技术平台并已经做了大量工作，筛选出有潜在价值的用于HCC诊断和预后的蛋白及发现新的治疗药物靶点，为HCC的诊断和治疗带来新的机遇。基质辅助激光解吸电离飞行时间质谱是蛋白质组学研究的核心技术之一。作者拟从MALDI—TOF-MS技术在肝细胞癌蛋白质组学研究中的应用方面作一综述。     

蛋白质芯片激光解析电离技术在肺癌研究中的应用

蛋白质芯片表面加强激光解析电离-飞行时间-质谱(SELDI-TOF-MS)技术是蛋白质组学研究的全新技术平台,进一步提高了蛋白质分离和鉴定的速度。并且在肺肿瘤生物标志物筛选、鉴别肺癌抗原和蛋白质指纹图谱方面取得突破性进展。今后该技术在肿瘤蛋白质组学研究中有更加广阔的应用前景。     

Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins

Previously, we demonstrated that a protein from Xenopus egg jelly exhibits sperm chemoattractant activity when assayed by either video microscopy or by sperm passage across a porous filter. Here we describe the isolation and purification of allurin, the protein responsible for this activity. Freshly oviposited jellied eggs were soaked in buffer, and the conditioned medium was loaded onto an anion exchange column and eluted with an NaCl gradient. The active fraction was purified further by RP-HPLC, the chemoattractant protein appearing as a single sharp peak. The amino acid sequence of the protein, determined by direct sequencing and cloning of cDNAs coding for the protein, consisted of 184 amino acids having a molecular mass of 21,073 Da. The protein shares homology with the mammalian cysteine-rich secretory protein (CRISP) family that includes testes-specific spermatocyte protein 1, a cell adhesion protein which links spermatocytes to Seritoli cells, and acidic epididymal glycoproteins that bind to sperm and have been implicated in sperm-egg fusion. Phylogenetic analysis suggests that allurin evolved from the ancestral protein that gave rise to the mammalian CRISP family. Addition of allurin to this family portends that the CRISP family represents a group of "sperm escort" proteins, which bind to sperm at various steps in their life history, facilitating passage from one functional stage to the next. Allurin stands out in this regard, representing both the first vertebrate sperm chemoattractant to be purified and sequenced and the first member of the CRISP family to be found in the female reproductive tract.     

Diagnosis of gastroenterological diseases by metabolome analysis using gas chromatography–mass spectrometry

Recently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis–mass spectrometry, and matrix assisted laser desorption ionization–mass spectrometry. Then, the findings of studies about GC–MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases.     

Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass

A glycosylphosphatidylinositol (GPI)-anchored hyaluronidase, PH-20, on the sperm surface has long been believed to assist sperm penetration through the cumulus mass surrounding the eggs. However, mouse sperm lacking PH-20 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus cells. Intriguingly, a 55-kDa hyaluronan-hydrolyzing protein was abundantly present in wild-type and PH-20-deficient mouse sperm. In this study, we purified the 55-kDa mouse protein from soluble protein extracts released from epididymal sperm by acrosome reaction and identified as a hyaluronidase, Hyal5. Hyal5 was exclusively expressed in the testis and formed a 160-kbp gene cluster together with Hyalp1, Hyal4, and Ph-20 on mouse chromosome 6. Hyal5 was a single-chain hyaluronidase present on the plasma and acrosomal membranes of sperm presumably as a GPI-anchored protein. Moreover, hyaluronan zymography revealed that Hyal5 is enzymatically active in the pH range 5-7 and inactive at pH 3 and 4. Both Hyal5-enriched PH-20-free soluble protein extracts and PH-20-deficient mouse sperm were capable of dispersing cumulus cells from the cumulus mass. Cumulus cell dispersal was strongly inhibited by the presence of a hyaluronidase inhibitor, apigenin. These results suggest that in the mouse, Hyal5 may function principally as a "cumulus matrix depolymerase" in the sperm penetration through the cumulus mass and in the local hyaluronan hydrolysis near or on the surface of the egg zona pellucida to enable the proximal region of sperm tail to move freely. PH-20 may compensate in part for the functional roles of Hyal5.     

F1Fo-ATPase, F-type proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding

The identification of host factors involved in virus replication is important to understand virus life cycles better. Accordingly, we sought host factors that interact with the influenza viral nonstructural protein 2 by using coimmunoprecipitation followed by mass spectrometry. Among proteins associating with nonstructural protein 2, we focused on the β subunit of the F1Fo-ATPase, which received a high probability score in our mass spectrometry analysis. The siRNA-mediated down-regulation of the β subunit of the F1Fo-ATPase reduced influenza virion formation and virus growth in cell culture. We further found that efficient influenza virion formation requires the ATPase activity of F1Fo-ATPase and that plasma membrane-associated, but not mitochondrial, F1Fo-ATPase is important for influenza virion formation and budding. Hence, our data identify plasma membrane-associated F1Fo-ATPase as a critical host factor for efficient influenza virus replication.     

The emergence of mass spectrometry in biochemical research.

The initial steps toward routinely applying mass spectrometry in the biochemical laboratory have been achieved. In the past, mass spectrometry was confined to the realm of small, relatively stable molecules; large or thermally labile molecules did not survive the desorption and ionization processes intact. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allow for the analysis of both small and large biomolecules through "mild" desorption and ionization methods. The use of ESI and MALDI mass spectrometry extends beyond simple characterization. Noncovalent interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery are among the areas to which ESI and MALDI mass spectrometry have been applied. This review summarizes recent developments and major contributions in mass spectrometry, focusing on the applications of MALDI and ESI mass spectrometry.     

Protein sequencing by tandem mass spectrometry.

Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.     

Whole body protein turnover in childhood Crohn''s disease.

In order to investigate the effects of treatment on protein metabolism in childhood Crohn's disease whole body protein turnover was measured using a primed constant intravenous infusion of L-[1-13C]leucine and mass spectrometry in 10 children with active disease. Mean rates of protein synthesis and breakdown markedly decreased after treatment with steroids (four) or an elemental diet (six). This suggests that protein synthesis and breakdown are increased during active Crohn's disease in children and are reduced after induction of remission regardless of the type of treatment.     

Dephosphorylation of a major sperm membrane protein is induced by egg jelly during sea urchin fertilization

A 160-kilodalton (kDa) protein of Arbacia punctulata sperm is constitutively phosphorylated on serine residues, as shown by in vivo³²PO₄^3- labeling. Exposure of sperm to solubilized egg jelly results in the immediate dephosphorylation (within 5 sec) of this protein and a simultaneous increase in its electrophoretic mobility (to an apparent molecular mass of 150 kDa). In its dephosphorylated form (150 kDa), the protein is a major component of the flagellar plasma membrane. In fact, silver-stained polyacrylamide gels show the 160/150-kDa protein to be the third most abundant protein in the whole flagellum. The spermatozoan must pass through the egg jelly layer on its way to the egg surface. The jelly-induced dephosphorylation of such an abundant sperm membrane protein may be an important event in the successful interaction of sperm and egg during fertilization.     

Antibiotics and probiotics in chronic pouchitis:A comparative proteomic approach

AIM:To profile protein expression in mucosal biopsies from patients with chronic refractory pouchitis following antibiotic or probiotic treatment,using a comparative proteomic approach. METHODS:Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry were used to characterize the changes related to antibiotic therapy in the protein expression profiles of biopsy samples from patients with chronic refractory pouchitis.The same proteom...     

Antibiotics and probiotics in chronic pouchitis:A comparative proteomic approach

AIM:To profile protein expression in mucosal biopsies from patients with chronic refractory pouchitis following antibiotic or probiotic treatment,using a comparative proteomic approach. METHODS:Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry were used to characterize the changes related to antibiotic therapy in the protein expression profiles of biopsy samples from patients with chronic refractory pouchitis.The same proteom...