Proteome analysis of apoptotic cells

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.     

Molecular pathways of oligodendrocyte apoptosis revealed by mutations in the proteolipid protein gene

A decade after the genetic link was established between mutations in the proteolipid protein gene and two leukodystrophies, Pelizaeus-Merzbacher disease and spastic paraplegia, the molecular mechanisms underlying pathogenesis are beginning to come to light. Data from animal models of these diseases suggest that the absence of proteolipid protein gene products in the central nervous system confers a relatively mild phenotype while missense mutations in and duplications of this gene give rise to mild or severe forms of disease. Previously, we have interpreted the disease process in terms of the accumulation of the mutant proteins in the secretory pathway and, herein, we review the evidence in favor of such a cellular mechanism. Furthermore, on the basis of recent data we suggest that the unfolded protein response may be involved in the pathogenesis of Pelizaeus-Merzbacher disease and spastic paraplegia through a kinase signaling cascade that links the accumulation of mutant proteins in the endoplasmic reticulum of oligodendrocytes with changes in gene regulation, protein synthesis, and possibly apoptosis.     

Crosslinking combined with mass spectrometry for structural proteomics

The method of crosslinking combined with mass spectrometry is being gradually accepted as a technology enabling detailed structural information on proteins and protein complexes. Intrinsic challenges of the method, which have prevented its widespread use, are being progressively addressed by improvements in mass spectrometry instrumentation capabilities, by the development of new crosslinking reagents, and by the development of specialized software tools for processing of mass spectrometric crosslinking data. This review focuses on recent literature concerning the development of specialized crosslinking reagents and approaches for mass spectrometry‐based applications. Critical features of crosslinking reagents for optimum mass spectrometric performance, such as isotopic coding, cleavability, affinity groups, structure of the linkers, and reactive groups, are assessed. Requirements for the design of crosslinking reagents to make them well suited for mass spectrometric detection and analysis are summarized. © 2010 Wiley Periodicals, Inc. Mass Spec Rev 29:862–876, 2010     

拟南芥外源ERG基因的阳性苗的初步筛选

近来关于Era蛋白(E.coli ras-like rotein)及其同源蛋白的研究较多,但其在真核乍物巾的功能仍不明朗,尤其是植物中相关研究报道尚少.拟南芥中有两个ERG蛋白,大小分别为437个氨基酸和427个氨基酸,分别用ERG437和ERG427表示.本文通过构建ERG基因的真核表达载体,利用农杆菌浸花法侵染拟南芥植株,筛选出阳性植株,为进一步定位观察和功能研究提供材料.     

我国大豆品质现状及其对策

对2002年、2003年全国大豆品种和品种资源粗蛋白、粗脂肪以及水溶性蛋白质普查数据进行了分析，结果表明：全国大豆品种粗蛋白、粗脂肪、水溶性蛋白平均含量分别为41，24％、19．78％，33.3％。其中大豆主产区品种粗蛋白、粗脂肪平均含量分别为39，97％、19．92％，大豆主产区品种的品质与全国品种品质水平接近。全国高蛋白、高油及双高品种使用率分别为8．7％、16．8％、11．2％。大豆品种资源粗蛋白、粗脂肪平均含量分别为41、77％、19.75％。变异系数分别在6．254％～7．510％、6，576％～9．232％。就目前大豆品种和品种资源品质现状，文中提出了提高大豆品种品质的建议及对策。     

Identification of protein associations in organelles, using mass spectrometry-based proteomics

Recent literature that highlights the power of using mass spectrometry (MS) for protein identification from preparations of highly purified organelles and other large subcellular structures is covered in this review with an emphasis on techniques that preserve the integrity of the functional protein complexes. Recent advances in distinguishing contaminant proteins from "bonafide" organelle-localized proteins and the affinity capture of protein complexes are reviewed, as well as bioinformatic strategies to predict protein organellar localization and to integrate protein-protein interaction maps obtained from MS-affinity capture methods with data obtained from other techniques. Those developments demonstrate that a revolution in cellular biology, fueled by technical advances in MS-based proteomic techniques, is well underway.     

短乳杆菌49蛋白提取及酶解条件优化

为了最大程度上获取短乳杆菌49（Lactobacillus brevis 49）的蛋白质信息，分别优化了蛋白质的提取条件与酶解条件。用正交实验法考察细胞破碎方法、蛋白酶种类、酶解时间、酶添加量4个因素对 L. brevis 49的胞内蛋白提取方法以及酶解方法的影响，以质谱中检测到的蛋白质数目为参考指标进行优化；同时对培养基氮源进行优化，并分别选用TCA沉淀法、冷丙酮沉淀法、平衡酚 丙酮法和超滤法提取发酵液中的胞外蛋白。结果表明：L. brevis 49胞内蛋白最佳的提取破壁方法为超声破碎法，最佳水解酶为糜蛋白酶和胰蛋白酶共同作用，最佳的酶与蛋白质量比为 1∶50，最佳酶解时间为12 h，获得胞内蛋白数目527个。获得L. brevis 49胞外蛋白的最佳培养基为0.6%酵母浸粉做单一氮源的MRS培养基，最佳提取方法为三氯乙酸（TCA）法，可获得44个含信号肽蛋白。该方法有望为啤酒的安全检测提供参考。     

Era蛋白（E.coli ras-like protein）-一个可能参与真、原细胞信号调控的新分子开关

充足的证据表明G蛋白作为真核细胞的重要分子开关参与细胞的增殖调控以及部分代谢调控过程。而近来在大肠杆菌中新发现的Era蛋白（E.coli ras-like protein）则是与已知的三聚体G蛋白和小分子G蛋白不同的一种新的GTP结合蛋白。进一步的研究发现该类GTP结合蛋白不仅存在于原核的大肠杆菌中,而且在高等植物、人类细胞中均含有该蛋白的同源蛋白。大肠杆菌的Era蛋白主要位于细胞膜的内侧,细胞质中也有一定的分布;一些证据表明,真核细胞ERA（ERG）蛋白来源于原核细胞,定位于线粒体或者叶绿体。近来的研究证据表明ERA或者ERG蛋白有可能担负着与其它两类G蛋白同样重要的分子开关功能。已有的研究表明Era蛋白参与调节原核生物的细胞分裂、细胞周期以及部分细胞代谢过程;在哺乳动物细胞中,同源蛋白ERA可能与细胞周期的G1期调控以及细胞凋亡有关;真核植物中相关研究报道尚少,推测该蛋白可能与种子的正常发育有关。本文主要介绍原核Era蛋白和真核ERA蛋白的结构特点以及功能研究进展。     

基质辅助激光解吸电离-串联飞行时间质谱鉴定鲨鱼硒结合蛋白

利用基质辅助激光解吸电离-串联飞行时间质谱(MALDI-TOF/TOF)高置信度地鉴定了原核表达的白鳍鲨硒结合蛋白。实验表明,硒结合蛋白肽段之一被错误指认为胰酶自水解峰和混合谱是影响其未进行串联质谱分析或不能被数据库搜索匹配的主要原因,同时还发现,虽然赖氨酸结尾肽段在MALDI源中信号强度普遍很低,但对它们进行串联质谱分析也可获得较好的谱图质量,部分数据可用于从头测序。考虑以上因素后,硒结合蛋白鉴定覆盖率由35%增至51%。     

行波离子迁移质谱技术在蛋白质结构研究中的应用

离子迁移(ion mobility)是一种在分子离子化后,非溶液环境下的分离技术。与依靠离子质量与电荷比进行分离的质谱技术不同,离子迁移是根据离子形状与电荷比实现分离的。因此,离子迁移技术可以实现对质量相同而形状不同的离子分离,其与质谱技术联用可为研究者提供更加丰富的结构信息。最新发展的行波离子迁移技术大大提高了灵敏度和分离速度,适用于研究天然浓度条件下的蛋白质,其在蛋白质单体折叠与去折叠以及蛋白复合体亚基组装的研究中已崭露头角。     

Modeling the clay minerals–enzyme binding by fusion fluorescent proteins and under atomic force microscope

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent–protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H–mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H–fusion enzymes, green–fusion enzyme showed higher intensity compared with other red and yellow fusion–proteins. Intensity of fusion–proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free‐state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme–soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals–enzyme complex.     

Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

For many years, amino acid‐specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI‐ or MALDI‐MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site‐specific structural information. In this review, we describe the reagents that are most commonly used in these residue‐specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 28:785–815, 2009     

Protein‐based stable isotope probing (protein‐SIP) in functional metaproteomics

The community phenotype as the sum of molecular functions of organisms living in consortia strongly depends on interactions within these communities. Therefore, the analyses of the most significant molecules in terms of the phenotype, the proteins, have to be performed on samples without disrupting the meta‐species environment. Due to the increasing genomic information, proteins provide insights into a potential molecular function and the phylogenetic structure of the community. Unfortunately, the lists of identified proteins are often based first on the technical capacity of the used methods or instruments, and second on the interpretation of them by the assignment of molecular functions to proteins in databases. Especially in non‐model organisms the functions of many proteins are often not known and an increasing number of studies indicate a significant amount of uncertainty. To decrease the dependency on assumptions and to enable functional insights by metaproteome approaches, the metabolic labeling from an isotopically labeled substrate can be used. Since the metabolites deriving from the substrate are very rarely species‐specific, the incorporation of the stable isotope into proteins can be used as a surrogate marker for metabolic activity. The degree of incorporation can be determined accurately on the peptide level by mass spectrometry; additionally, the peptide sequence provides information on the metabolic active species. Thereby, protein‐stable isotope probing (protein‐SIP) adds functional information to metaproteome approaches. The classical metaproteome approaches will be reviewed with an emphasis on their attempts towards functional interpretation. The gain from functional insights into metaproteomics by using metabolic labeling of stable isotopes of carbon, nitrogen, and sulfur is reviewed with a focus on the techniques of measurement, calculation of incorporation and data processing. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:683–697, 2012     

Study of the spinal cords of the sturgeon Acipenser schrenckii, gar Lepisosteus oculatus, and goldfish Carassius auratus by morphological, immunohistochemical, and biochemical approaches

Little is known about the spinal cords of phylogenetically ancient actinopterygeans. The spinal cords of the chondrostean Acipenser schrenckii (Amur sturgeon), holostean Lepisosteus oculatus (spotted gar), and teleost Carassius auratus (goldfish) were, therefore, analyzed by immunohistochemistry, electron microscopy and two-dimensional gel electrophoresis. Morphology showed numerous similarities between sturgeons and gars. In both, a dorsal column between the two dorsal horns was lacking, giving the grey matter an inverted Y-shape. In goldfish, a small dorsal column was seen, the grey matter occupied a larger area, neuronal density was much higher, and a ventral commissure was apparent, which was absent in sturgeons and gars. In the white matter of sturgeons and gars, small caliber axons predominated, whereas larger axons were frequent in goldfish. Choline acetyltransferase immunoreactive neurons were prevalent in the ventral horns of all three fish, mainly in motoneurons, but stained fibers were only found in sturgeons and gars. gamma-aminobutyric acid positive cells were seen in both the ventral and the dorsal horns of all three fish. Distribution of serotonin (5-HT) and tyrosine hydroxylase (TH) immunoreaction was similar in sturgeons and gars, being located in both the ventral and the dorsal horns. In goldfish, 5-HT label was confined to the ventral horn and TH label was mainly observed in a cell group located ventromedially. Two-dimensional gel electrophoresis showed a gradual increase in protein number from sturgeons to gars to goldfish. In conclusion, the spinal cords of sturgeons and gars share many morphological and chemical features, distinguishing them from the goldfish spinal cord.