Proteome-Scale Analysis of Biochemical Activity

ABSTRACT

During the last 10 years, there has been a large increase in the number of genome sequences available for study, altering the way that the biology of organisms is studied. In particular, scientific attention has increasingly focused on the proteome, and specifically on the role of all the proteins encoded by the genome. We focus here on several aspects of this problem. We describe several technologies in widespread use to clone genes on a genome-wide scale, and to express and purify the proteins encoded by these genes. We also describe a number of methods that have been developed to analyze various biochemical properties of the proteins, with attention to the methodology and the limitations of the approaches, followed by a look at possible developments in the next decade.     

Role of oncoproteomics in the personalized management of cancer

Oncoproteomics is the term used to describe the application of proteomic technologies in oncology and parallels the related field of oncogenomics. It is now contributing to the development of personalized management of cancer. Proteomic technologies are used for the identification of biomarkers in cancer, which will facilitate the integration of diagnosis and therapy of cancer. Molecular diagnostics, laser capture microdissection and protein biochips are among the technologies that are having an important impact on oncoproteomics. The discovery of protein patterns developed by the US Food and Drug Administration/National Cancer Institute Clinical Proteomics Program is capable of distinguishing cancer and disease-free states with high sensitivity and specificity and will also facilitate the development of personalized therapy of cancer. Examples of application are given for breast and prostate cancer and a selection of companies and their collaborations that are developing application of proteomics to personalized treatment of cancer are discussed. Continued refinement of techniques and methods to determine the abundance and status of proteins in vivo holds great promise for the future study of normal cells and the pathology of associated neoplasms. Personalized cancer therapy is expected to be in the clinic by the end of the first decade of the 21st century.     

Bulked sample analysis in genetics,genomics and crop improvement

Biological assay has been based on analysis of all individuals collected from sample populations. Bulked sample analysis (BSA), which works with selected and pooled individuals, has been extensively used in gene mapping through bulked segregant analysis with biparental populations, mapping by sequencing with major gene mutants and pooled genomewide association study using extreme variants. Compared to conventional entire population analysis, BSA significantly reduces the scale and cost by simplifying the procedure. The bulks can be built by selection of extremes or representative samples from any populations and all types of segregants and variants that represent wide ranges of phenotypic variation for the target trait. Methods and procedures for sampling, bulking and multiplexing are described. The samples can be analysed using individual markers, microarrays and high‐throughput sequencing at all levels of DNA, RNA and protein. The power of BSA is affected by population size, selection of extreme individuals, sequencing strategies, genetic architecture of the trait and marker density. BSA will facilitate plant breeding through development of diagnostic and constitutive markers, agronomic genomics, marker‐assisted selection and selective phenotyping. Applications of BSA in genetics, genomics and crop improvement are discussed with their future perspectives.     

绝经对血液单核细胞基因表达谱的影响:基因芯片初步研究

绝经是女性一生中很重要的生理现象之一,它能增加一系列复杂免疫、神经退化、新陈代谢和心血管方面的疾病。血液单核细胞能分化成各种各样的细胞,这些细胞在组织形态发生和免疫应答方面起着很重要的作用。本研究中采用了包含大约14,500个基因探针的Affymetrix Human U133A基因芯片来研究健康的绝经前和绝经后女性外周血液单核细胞中的基因表达谱。样本之间的对比分析表明有20个基因上调,20个基因下调。其中的28个基因根据它们的生物过程如细胞繁殖、免疫应答、细胞代谢等等被分成了6个主要的GO类别;剩下的12个基因其生物学功能还没有被鉴定。研究结果支持了我们的假设:血液单核细胞的功能状态确实受到绝经的影响,而且由此带来的改变可能是由全基因组范围的基因表达谱而决定的。本研究中鉴定的一些差异表达基因有可能作为以后研究与绝经相关的系统免疫、神经退化和心血管疾病的候选基因研究。此工作是这个研究方向的第一次尝试,为将来的进一步研究奠定了基础。     

Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies

The present work shows how UV 'light-induced molecular immobilisation' (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using 'LIMI,' arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1 microm in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies.