家蚕核多角体病毒的多角体蛋白基因

本文用AcMNPV多角体蛋白质基因的克隆DNA作探针,从BmS NPV DNASal Ⅰ酶酶解片段基因库中筛选出一个有同源顺序的克隆pBN 61。该克隆含大小为1.65 kb插入DNA片段。本文报道了该片段的Hind Ⅲ,Hpa Ⅱ和Alu Ⅰ等限制性内切核酸酶酶切图谱分析和部分DNA顺序分析结果。测得的DNA顺序中有138个碱基与已报道的BmSNPV多角体蛋白质一级结构中的46个氨基酸顺序相比较,除一个氨基酸发生变化外,其余完全相一致。克隆pBN 61可能包含完整的Bm SNPV多角体蛋白质结构基因。     

蛋白质顺序测定技术(一)

引言自从1955年F.Sanger测定了胰岛素结构以后,促进了蛋白质一级结构测定技术的发展。目前世界上搞清一级结构的蛋白质已有一千多个,其中最大的为β-半乳糖苷酶(β-Galactosidase),它由1021个氨基酸组成。氨基酸自动分析仪,自动顺序仪和高效液相层析技术的出现,更使蛋白质一级结构测定技术突飞猛进。目前仅须几个毫克的样品就能进行一级结构的测定。在我国,蛋白质一级结构的研究比较落后,但目前已有很多单位开展蛋白质一级结构的研究。为了更快促进我们这方面工作的进展,1980年10月西德马-普学会分子遗传研究所的B.Wittmann-Liebold     

赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估

采用胰蛋白酶(Trypsin)单独酶切与不同酶量的赖氨酸C端内切酶(Lys-C/trypsin)顺序酶切两种方法,对293T细胞全蛋白样本进行酶解消化,系统评估Lys-C/trypsin顺序酶切与Trypsin单一酶切在蛋白质组学样本制备中的差别.实验结果表明,Lys-C/trypsin顺序酶切不仅能显著提高肽段和蛋白质的鉴定数目,同时降低遗漏K酶切位点的数目及比例,而且得到的肽段长度有利于质谱鉴定,蛋白质覆盖率明显提升.通过对酶的用量进行优化对比,最终确定了Lys-C/trypsin顺序酶切时酶的合理用量.本研究结果对提高蛋白质组学样本的制备质量以及蛋白质的序列鉴定覆盖度具有指导意义.     

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

计算机辅助的羧肽酶法测定多肽羧端顺序

在初步用计算机辅助的羧肽酶法测定了天花粉蛋白C-端顺序的基础上, 进一步设计了两个计算机程序-DPS程序和CPA程序. 用合成小肽和天然的肽对这两个计算机程序进行模型实验证明, 运用这两个程序能分别满意地从羧肽酶的酶解动力学曲线中获得重要的C-端顺序信息, 并测定了天花粉蛋白分子中未知肽段CB-3的C-端顺序为: -SerAlaSerAlaLeuHserOH, 这一顺序后来已经其他实验结果所证实. 本法不仅使C-端顺序测定延长至七个氨基酸, 而且还基本上解决了多肽或蛋白质含有多种多次重复氨基酸残基的C-端顺序测定.     

荧光探针在蛋白质研究中的应用

荧光探针技术是研究蛋白质在水溶液中构象的一种有效方法。利用它可以测定蛋白质分子的疏水微区内两基团的距离以及酶与底物结合过程中蛋白质构象的变化等。本文综述了荧光探针技术在蛋白质研究中的一些进展。     

天花粉蛋白一级结构的修正及不同产地天花粉蛋白的研究

胰蛋白酶酶解天花粉蛋白, 用高效液相色谱分离酶解肽段, 用顺序仪测定其有关肽段的顺序。用羧肽酶A, B, Y测定了天花粉蛋白C-端和天花粉蛋白溴化氰降解肽CB1的C-端顺序, 修正了我们1985年测定的天花粉蛋白一级结构, 证明天花粉蛋白由246(7)氨基酸残基所组成, 除C-端微观不均一外, 与Collins结果一致。同时比较了芜湖产天花粉蛋白一级结构与平湖产的天花粉蛋白一级结构, 没有发现两者的一级结构有差别。     

质谱技术在蛋白质、多肽化学中的应用

文中介绍了几种新的质谱技术: 快原子轰击质谱(fast atom bombardment-MS, 简称FAB-MS)、串联质谱(tandem mass spectrometry, 简称MS/MS)、电喷雾电离质谱(electrospray ionization-MS, 简称ESI-MS)和基质辅助的激光解吸电离飞行时间质谱(matrix assisted laser desorption ionization time offlight MS, 简称MALDI-TOF-MS), 这几种技术的相互补充使得质谱比较有效地用于蛋白质结构测定。文中例举了几个实例说明了它们在蛋白质的分子量测定,蛋白质和多肽的纯度鉴定, 糖肽的结构测定及特殊的N-端封闭的或一般的蛋白质和多肽的顺序测定中的应用。     

Thin layer chromatography of dansyl and dinitrophenyl derivatives of amino acids. A review

The dinitrophenyl (DNP) derivatives of amino acids have found continual application in protein sequencing since Sanger used them for the first time for the sequencing of insulin. Dansyl derivatives of amino acids have been widely used in protein sequencing because of their fluorescent nature. The success of protein sequencing largely depends upon correct identification of such derivatives. The choice for the method of identification is related to cost, the availability of instrumentation and to the sensitivity needed for the analysis. Thin layer chromatography (TLC) is simple and has several advantages over other chromatographic methods. Therefore the literature after 1972 is reviewed for TLC analysis of dansyl- and DNP-amino acids, the two important amino acid derivatives required for identifying protein sequences. Additionally, the literature on the TLC resolution of enantiomeric mixtures of dansyl amino acids is reviewed. Application of various adsorbents, composition of solvent systems and other experimental conditions together with successful resolution data have been discussed. TLC provides a direct and inexpensive method for the resolution of enantiomers, and is fast becoming a sensitive instrumentalized quantitative analytical technique.     

Technical improvements in protein microsequencing

Improved techniques for preparing samples and sequencing reagents have been developed to achieve highly sensitive protein sequencing. These improvements include a new sequencing buffer system which gives extremely low chromatographic background, a method for preparing polybrene, a key chemical in gas-phase protein microsequencing, and techniques for preparing protein samples suitable for microsequencing.     

Nucleic Acid‐Barcoding Technologies: Converting DNA Sequencing into a Broad‐Spectrum Molecular Counter

The emergence of high‐throughput DNA sequencing technologies sparked a revolution in the field of genomics that has rippled into many branches of the life and physical sciences. The remarkable sensitivity, specificity, throughput, and multiplexing capacity that are inherent to parallel DNA sequencing have since motivated its use as a broad‐spectrum molecular counter. A key aspect of extrapolating DNA sequencing to non‐traditional applications is the need to append nucleic‐acid barcodes to entities of interest. In this review, we describe the chemical and biochemical approaches that have enabled nucleic‐acid barcoding of proteinaceous and non‐proteinaceous materials and provide examples of downstream technologies that have been made possible by DNA‐encoded molecules. As commercially available high‐throughput sequencers were first released less than 15 years ago, we believe related applications will continue to mature and close by proposing new frontiers to support this assertion.     

"Mass defect" tags for biomolecular mass spectrometry

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.     

质谱技术在免疫分子的结构研究中的应用

质谱技术用于生物大分子的研究具有直接、简单、快速、经济等优点。近十年来 ,基质辅助激光解吸质谱 (MALDI MS)和电喷雾质谱 (ESI MS)在免疫学领域的研究中作出了重要贡献。本文着重对抗原、抗体、抗原 抗体复合物、抗原决定簇等免疫分子结构的质谱研究作一评述。大体分为四方面内容 :免疫分子的分子量、翻译后修饰、异质性、构象变化的分析 ;质谱指纹图的取得和串联质谱测序 ;抗原 抗体复合物的证明 ;B 细胞表位和T 细胞表位序列的测定。这些研究结果对于理解免疫分子的免疫功能、对于疾病的早期诊断、对于发展新药和疫苗具有重要意义     

Escherichia coli single-stranded DNA-binding protein,a molecular tool for improved sequence quality in pyrosequencing

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application

The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone''s size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies.

A new technique for single molecular peptide sequencing is demonstrated by translocation of ssDNA-conjugated-peptide through MspA nanopore which is regulated by a DNA helicase motor.     

From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part I 1965-1984

As the title implies, the author undertakes a personal retrospective on the developments that since 1965 have shaped MS and taken it from a position of simply playing a role in protein chemistry to becoming an indispensable tool in proteomics, all in the past 40-year span. The article reviews the MS timeline of events, stopping at various time points where MS made significant contributions to protein chemistry or where the development of new instrumentation for MS represented a major advance for peptide and protein work. Major highlights in the field and their significance for peptide and protein characterization are covered, starting from the pioneering work carried out in the 1960s on peptide derivative formation and sequencing with instrumentation proper of that time, to later work done with triple, quad, and four-sector instruments, and on to the more recent work on the characterization of the proteome with ion traps, time-of-flight (TOF) instruments, and new ionization and tagging techniques.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.