ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Changes in proteomics profile during maturation of marrow-derived dendritic cells treated with oxidized low-density lipoprotein

Increasing evidence suggests that dendritic cells (DCs) and oxidized low-density lipoprotein (ox-LDL) participate in atherosclerosis. However, few data on the molecular mechanisms of this process are available. To address this question, we used iTRAQ labeling followed by LC-MS/MS analysis to identify many proteins that changed markedly during the maturation of dendritic cells stimulated with ox-LDL. Among a total of 781 identified proteins, 93 were upregulated and 100 were downregulated. The major and significant changes in upregulated proteins were that ox-LDL not only affected the levels of intracellular cathepsins G, Z, D and S, but also significantly enhanced cathepsin S secretion by the treated cells. Our results may provide clues for a more comprehensive understanding the pathogenesis of atherosclerosis.     

MicroRNAs Involved in Skeletal Muscle Differentiation

MicroRNAs (miRNAs) negatively regulate gene expression by promoting degradation of target mRNAs or inhibiting their translation. Previous studies have expanded our understanding that miRNAs play an important role in myogenesis and have a big impact on muscle mass, muscle fiber type and muscle-related diseases. The muscle-specific miRNAs, miR-206, miR-1 and miR-133, are among the most studied and best characterized miRNAs in skeletal muscle differentiation. They have a profound influence on multiple muscle differ-entiation processes, such as alternative splicing, DNA synthesis, and cell apoptosis. Many non-muscle-specific miRNAs are also required for the differentiation of muscle through interaction with myogenic factors. Studying the regulatory mechanisms of these miRNAs in muscle differentiation will extend our knowledge of miRNAs in muscle biology and will improve our understanding of the myogenesis regulation.     

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

人降钙素基因相关肽转基因马铃薯的RT-PCR分析

报道经过农杆菌介导将人降钙素基因相关肽(calcitoningenerelatedpeptide,CGRP)基因由马铃薯块茎专一表达classIpatatin基因5′侧翼区和CaMV35S启动子驱动构建的马铃薯表达载体导入马铃薯,PCR鉴定获得了转基因植株。RTPCR分析证实classIpatatin基因5′侧翼区驱动的CGRPmRNA在转基因马铃薯中的表达。研究结果在开发转基因马铃薯生物反应器表达医用多肽中具有重要意义。     

显脉獐牙菜的单萜环烯醚甙

近年来,肝炎发病率高,青叶胆(Swertia mileensis)因此被大量采挖,野生青叶胆资源逐年减少。为发掘新药源,我们对云南产狭叶獐牙菜(S.angustifolia)和显脉獐牙菜(S.nervasa)的化学成分进行了研究。本文报道从中分离鉴定的4个单萜环烯醚甙Ⅰ—Ⅳ。Ⅰ、Ⅲ、Ⅳ分别为维哥罗甙(vegeloside)、獐牙菜甙(sweroside)和獐牙菜苦甙(swertiamarin),Ⅱ是Ⅰ的同分异构体,命名为显脉獐牙菜甙(nervoside)。显脉獐牙菜甙(Ⅱ)白色无定形粉末,味极苦。UVλ-_(max)~(EtOH):243nm(log ε-3.89)。IR v_(max)~(KBr) cm~(-1):3400、1700和1610,显示单萜环烯醚的特征吸收峰显脉獐牙菜;显脉獐牙菜甙;单萜环烯醚甙