ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

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Highlights

• •ProAlanase is a powerful protease for efficient low pH disulfide bond mapping.
• •High suitability for analysis of histone family members and their PTMs.
• •Accurate phosphorylation profiling in proline-rich proteins.
• •Sequence coverage increase and full de novo sequencing in combination with trypsin.

     

一种简单高效的酵母单菌落 PCR 方法

目的:为快速简便地挑选出酿酒酵母重组克隆,探索建立一种经济、直接、高效的酵母单菌落 PCR 方法.方法:以 Leu2MX6基同重组或重组质粒转化得到的酵母突变菌为材料,分别采用传统的提取基组或质粒的方法、煮沸法及化学试剂处理法等制备 PCR 模板进行重组克隆鉴定,并对6种 PCR 模板制备方法的效果进行比较与分析;对加热提取法进行优化并进行重组子的提取和验证.结果与结论:直接以1 mm2单克隆菌株95℃处理5 min 后的酵母菌落水悬浮液为模板进行单菌落 PCR,是一种简单高效的酵母重组克隆鉴定方法.该方法能弥补传统方法的不足,且简便快速、结果稳定,可作为筛选和鉴定阳性克隆的有效手段.同时,这种单菌落 PCR 法也可应用重组毕赤酵母的阳性克隆筛选.     

病毒蛋白质组学及其应用

病毒蛋白质组学是蛋白质组学研究技术在病毒学领域的应用,其研究方法主要是基于质谱鉴定的电泳分离或色谱分离技术。病毒蛋白质组学的研究可以补充基因组注释、纯化单一的病毒成分、研究病毒与其宿主细胞蛋白的相互作用、识别病毒作用的靶位点、鉴定病毒感染的致病因子及病毒的进化关系、识别病毒的免疫源性蛋白。病毒蛋白质组的研究有助于对病毒致病性的了解,加速新的诊断方法及治疗药物的研制,增强对病毒的生物防御。由于一些技术及主观因素的影响,病毒蛋白质组的研究是很有限的,这是一个亟待重视并增强的领域。     

A Simple and Efficient Procedure for the Synthesis of an Alendronate- Oligonucleotide Conjugate via a Carbamate Linker

Abstract

In order to combine the biological properties of oligonucleotides, a synthetic chemical modelized reaction was performed and the procedure then applied to the preparation of an alendronate-deoxyoligonucleotide conjugate through a carbamate linker.     

一种简便高效的酵母基因组提取方法

提取基因组进行检测是酵母研究过程中的必要步骤之一。以毕赤酵母菌株GS115作为研究对象,主要成分为0.2 mol/L醋酸锂和1% SDS的酵母裂解液能高效的裂解酵母细胞壁。与两种酵母基因组提取试剂盒相比,该方法从相同体积的酵母培养液中获得的基因组的量高5倍以上,并且操作简便、快速,能在2 h内完成一次提取过程,极大地缩短了时间。以GS115中的内源AOX基因为目的基因,对提取的基因组进行PCR检测和Southern杂交检测,进一步验证了基因组的质量。因此,本文建立了一种简便、快速、经济而高效的酵母基因提取方法。     

定量蛋白质组学分析方法及应用

蛋白质组学经历了近10年的发展,现在已经初具规模。但是由于它是动态地观察生物体不断变化的所有蛋白质,所以技术难度非常之大。为使研究简化并更具针对性,人们着重进行比较蛋白质组学的研究。为了具体量化这些蛋白质的变化产生了定量蛋白质组学,近几年各种标记技术的进步使得该学科得以迅猛发展。     

一种简单高效的食用真菌总RNA提取方法

以金针菇为材料,建立了一种适合于富含RNase、多酚、多糖和糖蛋白的食用真菌RNA的提取方法,此方法在高浓度变性剂存在的条件下2次用苯酚-氯仿-异戊醇进行抽提去除DNA、蛋白质,并用异丙醇和乙酸钠选择性沉淀RNA、去除多糖,得到完整、均一的RNA样品。 Abstract:With Flammulina velutipes material,an improved method was developed for extracting total RNA from domestic fungus that are rich in RNase,polyphenols,polymeric carbohydrates and proteoglycans..Phenol-chloroform-isoamyl alcohol were used twice to clear DNA and protein under higher concentration of denaturing solution and isopentanol,sodium acetate were used to precipitate RNA selectively.Pure and intact RNA can be effectively prepared by this method.     

大规模合成长链RNA的简易低成本工艺

由于现有技术所限,RNA分子长度和二级结构往往成为RNA合成困难的主要原因.提供了一种简单低成本大规模制备和纯化长链RNA药物的新工艺,尤其针对具有稳定二级结构的长链RNA药物.采用引物延伸方法替代PCR和线性质粒DNA方法制备线性DNA模板可减少步骤及降低污染,然后用T7 RNA聚合酶转录制备的甲氧基修饰的线性DNA模板获得高均一度的长链RNA,转录粗产物直接用source 15Q阴离子HPLC分离T7 RNA聚合酶、rNTP、转录中断产物、内毒素和模板DNA等,从而获得高纯度RNA终产物.该工艺无需繁琐的酚/氯仿抽提和RNA变性,尤其适用于RNA的大量制备.     

A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering

Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.     

The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics,N-Glycomics and Glycosylation Site Assignment

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.N-glycosylation is one of the most abundant post-translational modifications of proteins. It is estimated that more than 50% of human proteins are N-glycosylated (1). This type of modification is critical to many fundamental biologic and pathologic processes such as: structural modulation of proteins, cell-cell signaling and interactions, pathogen-host recognition, and tumor progression (2, 3). Inherently, N-glycans are extremely heterogeneous, and subtle variations in the composition or structure may induce dramatic biological consequences (4, 5). Because of their heterogeneity, N-glycans typically need to be released from the parent glycoproteins to be accurately characterized or quantified (3, 6).Identifying the sugar-modified position (glycosite) in a glycoprotein is also critical to understanding the biological role of N-glycosylation (7). Current methods that determine glycosites often use sugar-based enrichment techniques, such as hydrazide chemistry (8) or lectin affinity (9). The extracted glycoproteins or glycopeptides are subjected to de-N-glycosylation, and the deglycosylated peptides are then sequenced by liquid chromatography-tandem MS (LC-MS/MS) to characterize the previously glycosylated sites with a standard bottom-up proteomic approach (10). Filtration has been previously applied to collect peptides (FASP) (11) and deglycosylated peptides after lectin-enrichment (N-glyco FASP) (12). Although these enrichment techniques can identify low-abundant glycosites because of the enrichment selectivity (13), typically they are not feasible for characterization of the N-glycome, because glycans are oxidized and altered when coupled to the hydrazide groups (8), and the selective affinity of the lectin usually biases the N-glycome.N-glycosylation is often considered “irreversible” once a glycoprotein is exported into the extracellular matrix. The addition of a dolichol-linked N-glycan precursor (Glc₃Man₉GlcNAc₂) onto a nascent peptide is an enzyme-catalyzed and nontemplate driven process (2). However, the likelihood and efficiency of this addition are impacted by many factors including: (1) the concentration and activity of oligosaccharyltransferase (2, 14), (2) the availability of dolichol-linked N-glycan precursor (2, 14), (3) the length of time the glycosylated region is unfolded during passage across the endoplasmic reticulum membrane (2, 14), (4) the accessibility of the glycosite, which is greatly impacted by neighboring amino acids (15), and (5) the conformation of a glycosylated protein (correctly folded or not) (14). Because of these variables, the majority of the common N-glycosylation consensus motif (Asn-XXX-Ser/Thr, in which XXX is any amino acid except proline) in human proteins are not actually modified by a sugar chain (15).Furthermore, a glycosite may be partially occupied (PO),¹ a state in which both the glycosylated (sugar-modified asparagine) and nonglycosylated (nonsugar-modified asparagine) forms coexist. For example, human corticosteroid-binding globulin, a major plasma glycoprotein with six N-glycosites, has variable degrees in occupancy among its six glycosites (ranging from 70 to 99.5%) that also seem to change with pregnancy (16). Although the biological implications of partial occupancy in N-glycosylation are not well understood, to date, there are no well-defined strategies that can readily identify PO glycosites, particularly in a complex mixture. Current sugar-based enrichment methodologies alone are typically incapable of determining whether a particular glycosite is partially occupied or not, because the nonglycosylated peptides are typically removed.Here we demonstrate a simple, rapid but comprehensive sample preparation platform, the GlycoFilter, which collects N-glycans and peptides separately in a single spin filter device. We demonstrate that glycans, including large acidic glycans, can be effectively separated and captured using a simple shift in pH combined with filtration. Although the lectin-based enrichment method of N-glyco FASP also uses a filtration principle to identify lectin-specific glycosites (12), this platform enables efficient downstream characterization of the N-glycans, N-glycosites, and the remaining proteome of a simple or complex biological sample. Furthermore, the GlycoFilter has the additional nonbiased capability to identify PO N-glycosites using a standard LC-MS/MS approach.     

化学蛋白质组学及其在新药开发中的应用

人类基因组计划提供的大量信息,很可能会掀起一场方法学上的革命,加速研究复杂生物体系的进程.但如何充分利用这些信息,从中挖掘出更好的方法,用于人类疾病的治疗?化学蛋白质组学的出现,提供了一种新方法,便于信息的提取,将加速从有效靶点建立到新药发现的进程.     

一种简单有效且适于土壤微生物多样性分析的DNA提取方法

参照Zhou[11]的方法进行了改进,获得了一种简单、有效的DNA提取方法.此方法操作简单、从大量样品改为小量样品的提取,利用高浓度的PEG沉淀,不作回收纯化,所提DNA片段较大,在23 kb以上,每克土的DNA提取量从3.74～15.28 μg,OD260/OD230比值在0.89～1.21范围内,用真菌和细菌核糖体特异性引物进行PCR扩增,均获得较好的结果,DGGE图谱显示丰富性较高,可用于细菌多样性和真菌多样性的分析.此方法能够从4种不同性质土壤中提取出DNA,但提取盐渍土壤和碱性土壤的效果更好一些,为土壤微生物群落结构的多样性分析奠定良好的基础.     

A Simple and Effective Approach for Digitization of the CTG Signals from CTG Traces

ObjectivesCardiotocography (CTG) is a useful tool for monitoring of the fetal heart rate (FHR) and uterine contractions (UC) during the intrauterine life. Generally, CTG is provided on a printed paper which is hard to save for future evaluations. So, digitization of CTG signals is in demand for future evaluations. A straightforward approach for digitization of the CTG signals is to apply image processing on the scanned CTG printed papers.Material and methodsIn this paper, an automatic procedure is proposed for digitization of the CTG signals. The proposed approach consists of four main stages such as pre-processing, image segmentation, signal extraction and signal calibration. The pre-processing stage covers median filtering and contrasts limited adaptive histogram equalization (CLAHE) for noise removal and contrast enhancement. Image segmentation is used to binarize the CTG images for signal determination using the Otsu's thresholding algorithm. The signal extraction is carried out by a two-stepped algorithm. The acquired CTG signals are then calibrated for obtaining the final CTG signals. We use the correlation coefficient to measure the similarity between the automatically digitized CTG signals and original signals.ResultsIn experimental works, an open-access database, which contains 552 CTG recordings, is employed. The results are quite impressive. According to the obtained results, the average correlation coefficients for FHR and UC signals are 0.9715 ± 0.0168 and 0.9717 ± 0.0465, respectively.ConclusionsThe obtained results show that the proposed method is quite efficient in digitization of the CTG signals. In future works, this tool will be used to digitize the recordings belonging to the antepartum period collected from the obstetrics clinics in Medical Park Hospital in Elazığ, Turkey.