单染色体靶向富集方法的建立

靶向富集技术具有良好的均一性、特异性和灵敏性,只需较低的测序覆盖度即可满足后续的分析。该技术被广泛应用于特定位点重测序和染色体构象等研究。为了靶向富集测序文库中特定的染色体DNA片段,本研究以21号染色体为例,采用人胚肝二倍体细胞CCC-HEL-1建立了一种单染色体靶向富集方法。具体步骤如下：（1）首先将人正常核型二倍体细胞同步化到G2/M 期,制备染色体悬液;然后流式分选出21号单染色体。（2）采用多重退火环形扩增技术（MALBAC）对21号染色体DNA进行扩增;用扩增的DNA产物构建文库。（3）利用体外转录合成、生物素标记的RNA探针与文库进行液相杂交,捕获并验证捕获效率。荧光定量PCR检测结果证明,特定的21号染色体呈上百倍的富集,而靶标文库则富集了近60 倍。结果提示,特异单染色体靶向富集方法成功建立。该法不仅可实现任一单染色体的靶向富集,用于单条染色体功能组学研究,而且还可能用于查找特定疾病相关的染色体异常（如DNA突变、异位、重复及倒置等）,具有一定的实用意义。     

Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

Solubilization of cross-linked cells followed by chromatin shearing is essential for successful chromatin immunoprecipitation (ChIP). However, this task, typically accomplished by ultrasound treatment, may often become a pitfall of the process, due to inconsistent results obtained between different experiments under seemingly identical conditions. To address this issue we systematically studied ultrasound-mediated cell lysis and chromatin shearing, identified critical parameters of the process and formulated a generic strategy for rational optimization of ultrasound treatment. We also demonstrated that whereas ultrasound treatment required to shear chromatin to within a range of 100–400 bp typically degrades large proteins, a combination of brief sonication and benzonase digestion allows for the generation of similarly sized chromatin fragments while preserving the integrity of associated proteins. This approach should drastically improve ChIP efficiency for this class of proteins.     

Method for Enrichment of Rhodopseudomonas sphaeroides from Fresh Water

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 10⁴ by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1^F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GF_n, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF₉, 0.24 mM and 86.5; GF₇, 0.33 mM and 132; GF₆, 0.85 mM and 71.2; GF₅, 3.8 mM and 25.4; GF₄, 2.8 mM and 28.8; GF₃, (nystose) 16 mM and 0.8; GF₂ (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GF_n, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites.     

Environmental Control of Root Exudation of Low-Molecular Weight Organic Acids in Tropical Rainforests

The availability of phosphorus (P) can limit net primary production (NPP) in tropical rainforests growing on highly weathered soils. Although it is well known that plant roots release organic acids to acquire P from P-deficient soils, the importance of organic acid exudation in P-limited tropical rainforests has rarely been verified. Study sites were located in two tropical montane rainforests (a P-deficient older soil and a P-rich younger soil) and a tropical lowland rainforest on Mt. Kinabalu, Borneo to analyze environmental control of organic acid exudation with respect to soil P availability, tree genus, and NPP. We quantified root exudation of oxalic, citric, and malic acids using in situ methods in which live fine roots were placed in syringes containing nutrient solution. Exudation rates of organic acids were greatest in the P-deficient soil in the tropical montane rainforest. The carbon (C) fluxes of organic acid exudation in the P-deficient soil (0.7?mol?C?m^?2?month^?1) represented 16.6% of the aboveground NPP, which was greater than those in the P-rich soil (3.1%) and in the lowland rainforest (4.7%), which exhibited higher NPP. The exudation rates of organic acids increased with increasing root surface area and tip number. A shift in vegetation composition toward dominance by tree species exhibiting a larger root surface area might contribute to the higher organic acid exudation observed in P-deficient soil. Our results quantitatively showed that tree roots can release greater quantities of organic acids in response to P deficiency in tropical rainforests.     

一种简易高效的小麦叶片胞间液蛋白提取方法的建立

植物细胞间液中的蛋白在植物生长发育和抗病抗逆反应及其信号传导过程中发挥着重要作用,是植物学研究的新热点.由于细胞间液含量低,容易因组织细胞损伤性受到胞内成分污染,高纯度的细胞间液蛋白的提取相对困难,参考相关文献报道,建立了一种简易高效的小麦叶片细胞间液蛋白的提取方法.选取小麦品种“铭贤169”幼苗叶片为材料,用50mmol/L NaAc缓冲液充分浸润,负压处理20 min后,30 × g离心5 min除去叶片表面缓冲液,随后2 000×g离心15 min,收集小麦叶片胞间液.将获得的小麦叶片胞间液冷冻干燥后,进行SDS-PAGE分离分析.电泳胶图上显示出细胞间液提取物与叶片组织提取物的蛋白质组成有极显著差异,使用MALD-TOF/TOF MS技术分析SDS胶上的细胞间液蛋白条带,共鉴定到9种非高丰度或假定的蛋白,其中有两种是已报道过的植物细胞间液蛋白.试验结果表明,本方法简易高效,适用于小麦蛋白质组学研究中高纯度的细胞间液蛋白的提取.     

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.     

Two Groups of Low Molecular Weight Hydrophobic Proteins from Barley Endosperm

The fraction under 25 000 molecular weight from the chloroform-methanol2: 1 (v/v) extract of barley endosperm contains two differentgroups of hydrophobic proteins. One group consists of the fourmajor components of the low molecular weight fraction presentin hordein preparations (A-hordeins). It is shown that theiramino acid compositions are outside the range of typical prolaminsand that therefore their designation as A-hordeins is inappropriate.Their chemical characteristics closely resemble those of thewheat CM proteins, which are also salt-soluble and hydrophobic.Components of the second group have high isoelectric points(>pH 9.0), molecular weights in the range 10 000–16000, and amino acid compositions within the definition of prolamins.They seem to be equivalent to the low molecular weight gliadinsfrom wheat, so it is suggested that they be designated low molecularweight hordeins.     

一种快速提取组织外泌体的分离富集方法

本文提供一种快速提取组织外泌体的分离富集方法。通过将目标组织用机械法切碎,加入组织消化酶进行组织解离和过滤,将获得的组织细胞悬液依次进行差速离心、超离、尺寸排阻和超滤,实现组织高质量外泌体的富集纯化。组织解离方法比较试验中,采用组织消化酶解离组织得到的蛋白质含量更高,获得的外泌体组织来源的蛋白质污染小。富集小鼠心组织、小鼠肝组织、小鼠肾组织、人结肠癌组织、人乳腺癌组织和动脉粥样硬化组织的外泌体,并对其进行纳米粒径追踪和透射电镜观察。结果显示,外泌体的粒径均在30~150 nm内,结构清晰明确。对小鼠肝组织富集的外泌体进行蛋白质印迹分析。结果显示,阳性蛋白质标志物CD9、ALIX和CD63的表达,TSG101弱表达,阴性蛋白质标志物Calnexin无表达。本方法集合多种分离措施,能够达到分离纯化外泌体的作用,同时简化了分离组织外泌体的步骤,相对于其他方法,全程只需要4~5 h,节省了富集时间,所富集的外泌体纯度高、可溶性杂蛋白质污染小,实用性更加广泛。使用微量组织样本富集的外泌体即可满足后续纳米粒径追踪、蛋白质印迹、透射电镜和转录物组等分析。     

纳米材料在蛋白多肽富集研究中的应用进展

复杂样本中低丰度蛋白的检测是蛋白质组学发展的难点和瓶颈。发展快速富集方法是检测低丰度蛋白有效途径之一,近年来研究表明纳米材料对蛋白／多肽存在不同程度的富集作用。结合国内外文献报道,对纳米材料在蛋白／多肽富集中的应用概况加以评述。     

Analysis of Membrane-Enriched and High Molecular Weight Proteins in Leishmania infantum Promastigotes and Axenic Amastigotes

Membrane and high molecular weight (HMW) proteins tend to be underrepresented in proteome analyses. Here, we optimized a protocol designed for the extraction and purification of membranes from the protozoan parasite Leishmania using a combination of serial centrifugation and free-flow zone electrophoresis (ZE-FFE). We also enriched for Leishmania HMW proteins from total extracts using the Gelfree 8100 fractionation system. This allowed the study of expression of both membrane-enriched and HMW proteins in Leishmania infantum promastigotes and amastigotes. We identified 194 proteins with at least one transmembrane domain (TMD) and 171 HMW proteins (≥100 kDa) in the invertebrate promastigote stage and 66 proteins with at least one TMD and 154 HMW proteins in the mammalian amastigote stage. Several of the proteins identified in one of the stages are part of pathways consistent with the known biology of the parasite, with many proteins involved in lipid synthesis, numerous dynein heavy chains, and some surface antigen proteins 2 detected in the promastigote stage. Notably, some proteins involved in transport and proteolysis were detected either in promastigote or amastigote. The present study is using improved proteomic methods for studying membrane-enriched and HMW proteins helping to achieve a better understanding of the parasite life cycle.     

Enrichment Map: A Network-Based Method for Gene-Set Enrichment Visualization and Interpretation

Background

Gene-set enrichment analysis is a useful technique to help functionally characterize large gene lists, such as the results of gene expression experiments. This technique finds functionally coherent gene-sets, such as pathways, that are statistically over-represented in a given gene list. Ideally, the number of resulting sets is smaller than the number of genes in the list, thus simplifying interpretation. However, the increasing number and redundancy of gene-sets used by many current enrichment analysis software works against this ideal.

Principal Findings

To overcome gene-set redundancy and help in the interpretation of large gene lists, we developed “Enrichment Map”, a network-based visualization method for gene-set enrichment results. Gene-sets are organized in a network, where each set is a node and edges represent gene overlap between sets. Automated network layout groups related gene-sets into network clusters, enabling the user to quickly identify the major enriched functional themes and more easily interpret the enrichment results.

Conclusions

Enrichment Map is a significant advance in the interpretation of enrichment analysis. Any research project that generates a list of genes can take advantage of this visualization framework. Enrichment Map is implemented as a freely available and user friendly plug-in for the Cytoscape network visualization software (http://baderlab.org/Software/EnrichmentMap/).     

一种精子表面GPI锚定蛋白初步鉴定方法的建立

精子表面糖基磷脂酰肌醇（GPI）锚定蛋白(GPI-Aps)在精子不同阶段都发挥着关键作用.本研究介绍了嗜水气单胞菌毒素（aerolysin）作为生物探针来鉴定精子表面的GPI锚定蛋白.实验依次进行了嗜水气单胞菌培养、毒素提取纯化、多克隆抗体制备以及三明治蛋白印迹验证实验.凝胶蛋白分离实验显示,硫酸铵沉淀加Sephadex G-100层析柱纯化得到了分子质量为45 kD高纯度细菌毒素|蛋白印迹实验验证了制备的多克隆抗体的特异性|三明治蛋白印迹结果显示,嗜水气单胞菌毒素能识别出12条精子脂筏提取蛋白条带,其分子质量主要分布在15~130 kD之间|Alex488标记的免疫荧光实验显示,嗜水气单胞菌毒素识别的GPI锚定蛋白主要分布在精子头部和尾部.研究结果提示,嗜水气单胞菌毒素能够有效地识别精子表面的GPI锚定蛋白.     

重组人血清白蛋白生产工艺研究进展

人血清白蛋白是一种重要的临床药物 ,人源血浆白蛋白可能会传染病原体将逐步被重组白蛋白所代替。对用巴斯德毕赤酵母生产重组人血清白蛋白的表达系统、发酵方法、分离纯化技术以及重组白蛋白的理化及生理特性进行了综述。     

Affinity Enrichment and Characterization of Mucin Core-1 Type Glycopeptides from Bovine Serum

The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Galβ1–3GalNAcα (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > ∼850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis.Glycosylation is among the most prevalent post-translational modifications of proteins; it is estimated that over half of all proteins undergo glycosylation during their lifespan (1). Glycosylation of secreted proteins and the extracellular part of membrane proteins occurs in the endoplasmic reticulum and the contiguous Golgi complex. The side chains of Trp, Asn, and Thr/Ser residues can be modified, termed as C-, N-, and O-glycosylation, respectively (2, 3). In addition, O-glycosylation also occurs within the nucleus and the cytosol: a single GlcNAc residue modifies Ser and Thr residues. O-GlcNAc glycosylation fulfills a regulatory/signaling function similar to phosphorylation (4).From an analytical point of view, C-glycosylation is the simplest. A consensus sequence has been defined: WXXW where the first Trp is modified, and the modification, a Man moiety, readily survives sample preparation and mass spectrometric analysis, including collisional activation (5). Investigation of N-glycosylation is also facilitated by several factors. First, N-glycosylation again has a well defined consensus sequence: NX(S/T/C) where the middle amino acid cannot be Pro (6). Second, there is a universal core glycan structure: GlcNAc₂Man₃; and this core is conserved across species. Third, a specific endoglycosidase, peptide N-glycosidase F, has been identified. This enzyme cleaves the carbohydrate structure from the peptide, leaving behind a diagnostic sign: the Asn residue is hydrolyzed to Asp, inducing a mass shift of +1 Da. By contrast, analysis of O-glycosylation is hampered by a lack of (i) a consensus sequence, (ii) a universal core structure, and (iii) a universal endoglycosidase or gentle chemical hydrolysis method to facilitate analysis.Glycosylation shows a high degree of species and tissue specificity; the same site may be modified by a wide variety of different glycan structures, and unmodified variants of the protein may occur simultaneously (7–9). Disease and physiological changes also may alter the glycosylation pattern (10–12). The biological role(s) of glycosylation has been studied extensively (13–15), although such studies are seriously hampered by the difficulties of glycosylation analysis.Most secreted proteins are glycosylated; and thus, mammalian serum is rich in glycoproteins. On the other hand, O-linked glycoproteins represent a small percentage of the serum protein content. Glycoproteins may display a befuddling heterogeneity both in site specificity and site occupancy. Thus, the enrichment of modified proteins or peptides is necessary for their characterization, and different techniques have been tested for this purpose. Lectin affinity chromatography is a popular method for selective isolation of glycoproteins and glycopeptides. Concanavalin A can be used to isolate oligomannose type glycopeptides (16), wheat germ agglutinin is applied for GlcNAc-containing compounds (16, 17), and jacalin is selective for core-1 type O-glycopeptides (18, 19). Lectins with preferential affinity for fucosylated and sialylated structures can also be utilized (12). Non-selective capture of glycopeptides can be performed using hydrophilic interaction chromatography (20, 21) or size exclusion chromatography (22). A recent approach applies porous graphite columns for semiselective enrichment (23), whereas the acidic character of sialylated glycopeptides has also been exploited via titanium dioxide-mediated enrichment (24). Finally vicinal cis-diols can be selectively captured using boronic acid derivatives (25–27). All methods described here provide some glycopeptide enrichment from non-glycosylated peptide background, but all also suffer from significant non-selective binding. N-Linked glycoproteins may also be selectively captured on hydrazide resin following periodate oxidation (28). This approach requires enzymatic deglycosylation to release the captured peptides for analysis, therefore excluding the determination of the carbohydrate structure.Intact glycopeptide characterization still represents a significant challenge. Edman degradation, either alone or in combination with mass spectrometry, has been utilized for such tasks (29, 30). CID analysis of O-linked glycopeptides has limited utility. (i) MS/MS analysis cannot differentiate between the isomeric carbohydrate units and usually does not reveal the linkage positions and the configuration of the glycosidic bonds. (ii) Such spectra are typically dominated by abundant product ions associated with carbohydrate fragmentation, namely non-reducing end oxonium ions and product ions formed via sequential neutral losses of sugar residues from the precursor ions. (iii) The glycan is cleaved from the peptide via a gas-phase rearrangement reaction, and as a result the peptide itself and most peptide fragments (if any) are detected partially or completely deglycosylated (31–33). Recently a different approach, the combination of positive and negative ion mode infrared multiphoton dissociation, was found to provide conclusive structural assignment for some O-linked glycopeptides (34). However, two novel MS/MS techniques, electron capture dissociation (ECD),¹ which is performed in FT-ICR mass spectrometers (35), and electron transfer dissociation (ETD), which is performed in various ion trapping devices (36), may represent the real breakthrough. In both cases an electron is transferred to multiply protonated peptide cations, triggering peptide fragmentation at the covalent bond between the amino group and the α-carbon, producing mostly c and radical z· product ions while leaving the side chains intact. ETD is typically more efficient than ECD and thus leads to more comprehensive fragmentation. In addition, ETD can be performed in ion traps and thus, at a higher sensitivity level, especially in a linear ion trap. Because it has been observed that there are instances when the electron transfer is efficient and still no significant fragmentation occurs, ETD is usually combined with supplementary (and gentle) CID activation (37). O-Glycosylation analysis using these new dissociative techniques has been investigated (38, 39). However, because of the complexity of extracellular O-glycosylation, analysis of complex mixtures is rarely attempted (18), and the above techniques are usually restricted to the analysis of purified proteins.In this study we present the analysis of secreted O-linked glycopeptides. Lectin (jacalin) affinity chromatography was used to achieve some enrichment of core-1 O-GalNAcα type carbohydrate-carrying glycopeptides from bovine serum. The glycopeptide fractions were subjected to CID and ETD analysis. These experiments were performed on a linear ion trap-Orbitrap hybrid mass spectrometer (40). The Orbitrap delivered high resolution, high mass accuracy for the precursor ions, whereas the linear trap provided high sensitivity MS/MS analyses. Some fractions were also subjected to sequential exoglycosidase digestions, and glycopeptides retaining only the proximal GalNAc residues were analyzed. ProteinProspector v5.2.1, developed to accommodate ETD product ion spectra, aided data interpretation (41). We identified 26 glycosylation sites from bovine serum unambiguously; 21 of these sites have never been reported by any other study. No other single study to date has yielded so much information about O-linked glycosylation sites.     

Head Proteins from T-Even Bacteriophage: I. Molecular Weight Characterization 1

T-even bacteriophage capsid proteins were separated on 6% agarose columns by use of 6 m guanidine hydrochloride containing 5 mm dithiothreitol both to dissociate and to elute the proteins. The head capsids of T2H, T4B, T4B01, T4D, and T6r(+) contained at least three structural proteins with molecular weights of 40,000, 18,000, and 11,000 daltons, amounting to 76, 2, and 8%, respectively, of the total capsid protein. On the other hand, T2L head capsids contained only two structural proteins with molecular weights of 40,000 and 18,000 daltons (81 and 2.5%, respectively, of the total protein). A discussion of the possible role of these structural head proteins and a T-even phage head model suggesting a structural arrangement of the 40,000 dalton subunit are presented.