Annual Spring Meeting of the Proteomics Standards Initiative

The Human Proteome Organization Proteomics Standards Initiative has produced reporting requirements and data interchange formats for the proteomics community. The implementation of these increasingly mature formats was the main focus of this meeting, with extensions being made to many schema to enable encoding of new data types. The endorsement of the proteomics standards initiative standards by an increasing number of journals is a main driving force behind tool development and a recognized need to ease the process of data deposition into the public domain for the bench scientist.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

一种提高质粒DNA凝胶电泳检测灵敏度的方法

比较了凝胶电泳示检测质粒ＤＮＡ时不同激发波长对ＤＮＡ－ＥＢ荧光强度的影响,发现短波长激发光可增加ＤＮＡ的探测灵敏度。采用２６０ｎｍ作为激光光时可探测到少至０．７ｎｇ的线性ＤＮＡ。且在很广的ＤＮＡ的质量范围内,ＤＮＡ－ＥＢ荧光强度 与ＤＮＡ量或正比。以此改进方法检测电离辐射诱导的ＤＮＡ单、双链断裂岢得到与其它研究结果相一致的Ｇ（ＳＳＢ）和Ｇ（ＤＳＢ）值。     

The work of the Human Proteome Organisation's Proteomics Standards Initiative (HUPO PSI)

This article describes the origins, working practices and various development projects of the HUman Proteome Organisation's Proteomics Standards Initiative (HUPO PSI), specifically, our work on reporting requirements, data exchange formats and controlled vocabulary terms. We also offer our view of the two functional genomics projects in which the PSI plays a role (FuGE and FuGO), discussing their impact on our process and laying out the benefits we see as accruing, both to the PSI and to biomedical science as a whole as a result of their widespread acceptance.     

人脑胶质细胞瘤核染色质非组蛋白的聚丙烯酰胺凝胶电泳分析

用不连续SDS-聚丙烯酰胺凝胶电泳分析了人脑胶质细胞瘤与正常脑细胞核NHCP电泳图谱。从二者的NHCP电泳结果表明,脑胶质细胞瘤增加了一条表观分子量为3万的蛋白区带;表观分子量为1.75万～6.5万的蛋白区带染色明显加深。染色质紫外吸收光谱也有明显差异。总之,脑胶质细胞瘤核NHCP发生质与量的变化。     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

Annual spring meeting of the Proteomics Standards Initiative 23-25 April 2008, Toledo, Spain

The role of the Human Proteome Organisation Proteomics Standards Initiative (HUPO-PSI) is to produce and release community-accepted reporting requirements, interchange formats and controlled vocabularies for mass spectrometry proteomics and related technologies such as gel electrophoresis, column chromatography and molecular interactions. A number of significant advances were made at this workshop, with the new MS standard, mzML, being finalised prior to release on 1(st) June 2008 and analysisXML, which will allow protein and peptide identifications and post-translational modifications to be captured, being prepared to enter the review process this summer. The accompanying controlled vocabularies are continuing to evolve and a number of standards papers are now being finalised prior to publication.     

Getting a grip on proteomics data – Proteomics Data Collection (ProDaC)

In proteomics, rapid developments in instrumentation led to the acquisition of increasingly large data sets. Correspondingly, ProDaC was founded in 2006 as a Coordination Action project within the 6th European Union Framework Programme to support data sharing and community‐wide data collection. The objectives of ProDaC were the development of documentation and storage standards, setup of a standardized data submission pipeline and collection of data. Ending in March 2009, ProDaC has delivered a comprehensive toolbox of standards and computer programs to achieve these goals.     

Further steps in standardisation. Report of the second annual Proteomics Standards Initiative Spring Workshop (Siena, Italy 17-20th April 2005)

The spring workshop of the HUPO-PSI convened in Siena to further progress the data standards which are already making an impact on data exchange and deposition in the field of proteomics. Separate work groups pushed forward existing XML standards for the exchange of Molecular Interaction data (PSI-MI, MIF) and Mass Spectrometry data (PSI-MS, mzData) whilst significant progress was made on PSI-MS' mzIdent, which will allow the capture of data from analytical tools such as peak list search engines. A new focus for PSI (GPS, gel electrophoresis) was explored; as was the need for a common representation of protein modifications by all workers in the field of proteomics and beyond. All these efforts are contextualised by the work of the General Proteomics Standards workgroup; which in addition to the MIAPE reporting guidelines, is continually evolving an object model (PSI-OM) from which will be derived the general standard XML format for exchanging data between researchers, and for submission to repositories or journals.     

Visualisation and analysis of proteomic data from the procyclic form of Trypanosoma brucei

We have undertaken a large scale study of the proteins expressed in the procyclic form of the parasite Trypanosoma brucei, which causes African sleeping sickness, using 2-DE and MS. The complete data set encompasses over 2000 identifications, of which 770 are distinct proteins. We have discovered that multiple protein isoforms appear to be common in T. brucei, as most proteins have been matched to more than one gel spot. We have developed visualisation software to investigate the differences between isoforms, based on the information from the results of database searches with MS data. We are able to highlight instances where PTMs are the most likely cause of variant forms. In other cases, spots that appear reproducibly across replicates contain fragments of proteins, arising either as experimental artefacts or as part of protein degradation. We are also able to classify clusters of gel spots into different groups based on the pattern of peptides that have been matched from MS data. The entire data set is stored within a relational database system that allows complex queries ( http://www.gla.ac.uk/functionalgenomics). Using specific proteins as examples, we demonstrate how the visualisation software and the database query facilities can be used.     

变性梯度凝胶电泳在实验小鼠细菌检测中的应用

目的 研究变性梯度凝胶电泳(denatured gradient gel electrophoresis, DGGE)在实验小鼠细菌检测中的应用。方法 根据16S rDNA V3区引物,PCR扩增3种实验小鼠(KM小鼠、NIH小鼠和BALB/c小鼠)呼吸道和盲肠段的细菌基因组DNA;扩增产物运用DGGE进行电泳检测,并分析条带数量间差异的统计学意义。结果 KM小鼠盲肠段条带12~18条,呼吸道条带5~10条;NIH小鼠盲肠段条带15~20条,呼吸道条带4~10条;BALB/c小鼠盲肠段条带10~15条,呼吸道条带0~7条。统计分析结果显示,KM小鼠和NIH小鼠在盲肠和呼吸道电泳条带数量上的差异无统计学意义( P >0.05);BALB/c小鼠与KM小鼠、NIH小鼠间的差异均有统计学意义( P <0.05)。结论 DGGE 在实验小鼠盲肠和呼吸道细菌检测中能较好地反映菌群的物种多样性。     

From proteins to systems: the British Society for Proteomics Research (BSPR) meeting 2005

This report describes the highlights of the second scientific meeting of the British Society for Proteome Research (BSPR), jointly organised with the European Bioinformatics Institute (EBI), and held at The Genome Centre, Cambridge UK in July 2005. The theme of the meeting was "From Proteins to Systems" covering many diverse aspects of proteomics, bioinformatics and systems biology.     

An Epidemiological Study of Salmonella enteritidis by Pulsed-Field Gel Electrophoresis (PFGE): Several PFGE Patterns Observed in Isolates from a Food Poisoning Outbreak

An epidemiological analysis of Salmonella enteritidis from a food poisoning was done using pulsed-field gel electrophoresis (PFGE) of BlnI- or XbaI-digested fragments of chromosomal DNA of isolates. S. enteritidis isolates obtained from 19 patients had identical PFGE patterns. Therefore, a strain giving the same pattern was considered to be the causative agent of this outbreak. In addition, four isolates that had different BlnI-digested PFGE patterns were obtained from three patients, suggesting that the observed variations in PFGE patterns might occur as the result of some point mutations of chromosomal DNA during growth or from the existence of several S. enteritidis strains from various sources. Subsequent PFGE analysis of continuously subcultured strains supported the former possibility. These observations indicate that PFGE analysis on multiple numbers of colonies from each patient are necessary for the epidemiologic investigation of S. enteritidis.     

应用蝌蚪快速检测环境变异的两种方法--微核试验和单细胞凝胶电泳

本文介绍了应用无尾两栖类动物的蝌蚪进行环境监测的两种方法——微核试验和单细胞凝胶电泳。此两种环境检测的方法与其他环境检测方法相比具有快速,简便,易操作,适于检测现场应用,可大面积推广等优点。     

The HUPO pre-congress Proteomics Standards Initiative workshop. HUPO 5th annual World Congress. Long Beach, CA, USA 28 October-1 November 2006

The plenary session of the Proteomics Standards Initiative of the Human Proteome Organisation provided an opportunity to update delegates on the progress of the work of the Human Proteome Organisation's Proteomics Standards Initiative (HUPO-PSI) to develop and implement standards in the field of proteomics. Significant advances have been made since the previous congress, with several of the interchange standards and minimal requirements documents being submitted for publication in the literature and being more widely adopted by both manufacturers and data repositories. An exciting development over the interim twelve months is the ongoing merger of the two existing mass spectrometry standards, the PSI mzData and Institute for Systems Biology mzXML, into a single product. This should be achieved by early in 2007.     

用脉冲电场凝胶电泳分析棉病囊霉及其突变体的染色体DNA

采用交变脉冲电场凝胶电泳和碱变性交变脉冲电场凝胶电泳方法,分析了棉病囊霉酵母菌及其2个不同的突变菌株的核型,得知此菌株含有5条染色体 DNA,而2株突变体的染色体 DNA 都没有大片段的缺失或双链断裂,但其稳定性不如野生型菌株的 DNA,而且存在单链断裂等碱不稳定性位点.