MASS SPECTROMETRY IN VIROLOGICAL SCIENCES

Virology, as a branch of the life sciences, discovered mass spectrometry (MS) to be the pivotal tool around two decades ago. The technique unveiled the complex network of interactions between the living world of pro- and eukaryotes and viruses, which delivered “a piece of bad news wrapped in protein” as defined by Peter Medawar, Nobel Prize Laureate, in 1960. However, MS is constantly evolving, and novel approaches allow for a better understanding of interactions in this micro- and nanoworld. Currently, we can investigate the interplay between the virus and the cell by analyzing proteomes, interactomes, virus-cell interactions, and search for the compounds that build viral structures. In addition, by using MS, it is possible to look at the cell from the broader perspective and determine the role of viral infection on the scale of the organism, for example, monitoring the crosstalk between infected tissues and the immune system. In such a way, MS became one of the major tools for the modern virology, allowing us to see the infection in the context of the whole cell or the organism. © 2019 John Wiley & Sons Ltd. Mass Spec Rev     

Applications and advancements of FT-ICR-MS for interactome studies

The set of all intra- and intermolecular interactions, collectively known as the interactome, is currently an unmet challenge for any analytical method, but if measured, could provide unparalleled insight on molecular function in living systems. Developments and applications of chemical cross-linking and high-performance mass spectrometry technologies are beginning to reveal details on how proteins interact in cells and how protein conformations and interactions inside cells change with phenotype or during drug treatment or other perturbations. A major contributor to these advances is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) technology and its implementation with accurate mass measurements on cross-linked peptide-pair precursor and fragment ions to enable improved identification methods. However, these applications place increased demands on mass spectrometer performance in terms of high-resolution spectral acquisition rates for on-line MSⁿ experiments. Moreover, FT-ICR-MS also offers unique opportunities to develop and implement parallel ICR cells for multiplexed signal acquisition and the potential to greatly advance accurate mass acquisition rates for interactome studies. This review highlights our efforts to exploit accurate mass FT-ICR-MS technologies with chemical cross-linking and developments being pursued to realize parallel MS array capabilities that will further advance visualization of the interactome.     

非变性离子淌度质谱在蛋白质结构及构象疾病研究方面的应用

离子淌度质谱技术可以分离空间尺寸或构象不同的离子,能够在近似生理条件下表征蛋白质及其复合物的构象,可提供蛋白质及其复合物的构象稳定性及异质性、化学计量比等多重信息,已成为蛋白质构象及蛋白质-配体相互作用研究的重要手段。非变性离子淌度质谱还具有灵敏捕获蛋白质构象动态转变的特点,适用于低浓度、高异质性的蛋白混合物分析。本文综述了离子淌度质谱的基本原理、获取数据及信息形式、以及在蛋白质构象及蛋白质-配体相互作用研究领域的应用进展,重点关注其在蛋白质错误折叠、聚集动力学及与配体相互作用的应用研究。     

Monitoring of S100 homodimerization and heterodimeric interactions by the yeast two-hybrid system

The S100 family consists of 19 members, which function as transducers of calcium signals in a tissue-specific manner. Upon calcium binding, the conformation of many S100 proteins changes dramatically. Several hydrophobic residues are exposed, allowing the S100 proteins to interact with their target proteins, and thereby to transduce calcium signals into specific biological responses. To further elucidate the exact contribution of the S100 calciproteins in the calcium signalling pathways, several groups have applied the yeast two-hybrid technology to identify putative target proteins for the various S100 calciproteins. Two-hybrid large screens using S100 proteins as baits have confirmed the biochemical and structural feature of S100, which enable them to form homodimers and the ability of some members to form specific heterodimers in vivo. Yeast two-hybrid investigations have allowed the identification of conserved hydrophobic residues and domains that are crucial for the stabilization of S100 homo- and heterodimers. Furthermore, this method clearly underlines that the homo- and heterodimerization mechanisms differ among the members of the S100 family. However, several lines of evidence strongly suggest that two-hybrid methodology is limited to the analysis of interactions that are calcium-independent, since no target proteins other than S100 family members themselves have been detected with this methodology.     

Dephosphorylated mutations affect the protein-protein interactions of ERF in <i>Populus simonii x P. nigra</i>

Phosphorylation is a common type of post-translational modification (PTM). It plays a vital role in many cellular processes. The reversible phosphorylation and dephosphorylation affect protein structures and proteinprotein interactions. Previously, we obtained five proteins that interact with ethylene-responsive factor (ERF) from the cDNA library of Populus simonii x Populus nigra. To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability, we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay (Y2H). The secondary structures and 3D structures of PsnERF, ERFm, TrunERF, and psnerf^197/198/202a were predicted by homology modeling. The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions, while dephosphorylated mutations blocked the interactions. Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation, and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.     

AB SCIEX TripleTOF 5600在蛋白质组学研究中的应用

ABSCIEX公司最新发布了全新的高分辨液相色谱质谱联用系统，TripleTOF5600。这一新产品是在享有盏名的API5500三重四极杆系统和QSTAR Elite（QqTOF）系统这两个技术平台的基础上开发出来的。这一全新的TripleTOF5600高分辨液相色谱质谱联用系统能够对复杂的蛋白质组学样品进行高通量分析，并能同时提供高质量的定性和定量结果。为了评估这款仪器的性能，我们设计了基于纳升液相色谱和TripleTOF5600联用系统的一系列实验。首先，我们应用这一拥有高分辨率、高灵敏度和高质量准确度的系统对酵母细胞裂解物的胰酶消化产物进行了分析。实验中采用了每秒最多采集50幅串联质谱谱图的信息相关采集模式，单次LC-MS／MS实验就鉴定了10，179条独特肽段和1174种蛋白（at 1％global false discoveryrate）。而实际达到了平均每秒54幅串联谱图的采集速度是在单次实验获得如此优异的鉴定结果的决定性因素。此外，我们也对一组含有五种已知蛋白以及部分重同位素标记肽段的样品进行了分析，以检验Triple TOF 5600的定量能力。实验中这五种蛋白质都获得了很高的序列覆盖率，其中进样量仅有1OOamol的Peroxiredoxin1蛋白的序列覆盖率也高达55％。而测试中得到肽段的平均质量偏差仅为1．7ppm，展示了Triple TOF 5600的高质量准确度和稳定性。我们还获得了高度准确的定量结果，充分证明了TrIple TOF 5600能够提供三重四极杆系统级别的定量结果。     

AB SCIEX TripleTOF 5600在蛋白质组学研究中的应用

AB SCIEX公司最新发布全新的高分辨液相色谱质谱联用系统——TripleTOF5600是在享有盛名的API 5500三重四极杆系统和QSTAR Elite(QqTOF)系统这两个技术平台的基础上开发出来的。TripleTOF 5600高分辨液相色谱质谱联用系统能够对复杂的蛋白质组学样品进行高通量分析,并能同时提供高质量的定性和定量结果。为评估这款仪器的性能,设计基于纳升液相色谱和TripleTOF 5600联用系统的一系列实验。首先,应用这一拥有高分辨率、高灵敏度和高质量准确度的系统对酵母细胞裂解物的胰酶消化产物进行分析。实验中采用每秒最多采集50幅串联质谱谱图的信息相关采集模式,单次LC-MS/MS实验就鉴定10179条独特肽段和1174种蛋白(at 1%global false discoveryrate)。而实际达到平均每秒34幅串联谱图的采集速度是在单次实验获得如此优异的鉴定结果的决定性因素。此外,也对一组含有5种已知蛋白以及部分重同位素标记肽段的样品进行分析,以检验TripleTOF 5600的定量能力。实验中这5种蛋白质都获得很高的序列覆盖率,其中进样量仅有100amol的Peroxiredoxin 1蛋白的序列覆盖率也高达53%。而测试中得到肽段的平均质量偏差仅为1.7 ppm,展示TripleTOF 5600的高质量准确度和稳定性。此外还获得高度准确的定量结果,充分证明TripleTOF 5600能够提供三重四极杆系统级别的定量结果。     

全自动整体式毛细管规模液相色谱系统——Waters CapLC~(TM)系统

毛细管液相色谱系统是Waters多年心血的杰作。它利用最先进的光纤技术,使系统可以提供比传统分析规模HPLC高50倍的灵敏度。毛细管水平的流路不仅几乎无死体积,而且可以平稳而高度重现地输出低至1ul/min的流动相,非常适合与质谱检测器的连接。精心的设计与制作,使CapLC系统能够提供优于传统HPLC系统的保留时间重现性与分辨率。系统很适合分析样品量很小的被测物,在疾病目标识别、生化分子和小分子药物的药代动力学研究等方面有广阔的应用前景。 CapLC配以快速高吞吐量进样器和二极管矩阵检测器,是全自动的毛细管LC/MS系统组件     

非变性质谱相关技术的研究进展

蛋白质与其他分子的相互作用几乎在所有的生命活动中起着核心调控作用,这些相互作用力和形成的蛋白质复合物是现代生命科学的研究重点。由于传统的生物物理技术对蛋白质复合物和相互作用的研究存在样品纯度要求高的限制,因此迫切需要新技术的出现,为结构生物学和相互作用组学的研究提供补充。质谱技术可以从原理上对混合样品进行检测,降低对样品纯度的要求,其中非变性质谱展现出强大的连接与互补作用。本文从样品制备、离子源、质量分析器、质谱联用技术等4方面介绍非变性质谱相关技术及近年来的研究进展,并总结分析未来面临的挑战以及发展方向。     

液相色谱-串联质谱技术在临床检验中的应用研究进展

液相色谱-串联质谱(LC-MS/MS)技术具有高灵敏度、高特异性、高分辨率和高效率的优点。近年来随着仪器灵敏度的提高,LC-MS/MS在常规临床检验中显示出极大的潜力,并在疾病早期预防和诊断中发挥着不可替代的作用。本文对LC-MS/MS在新生儿疾病筛查、维生素D检测、内分泌激素检测、肽类和蛋白质定量分析等临床检验方面的研究进展进行综述,并讨论了未来面临的挑战。     

细胞膜色谱在线HPLC-IT-TOF MS联用系统筛选分析射干中次野鸢尾黄素的抗EGFR活性作用

中药射干具有多种药理作用并得到广泛研究,但对其抗肿瘤活性组分的研究较少。本研究建立了一种用于筛选射干中抗肿瘤活性组分的方法。利用高表达表皮生长因子受体(EGFR)细胞制备细胞膜色谱柱,并通过十通切换阀与高效液相色谱-离子阱-飞行时间质谱(HPLC-IT-TOF MS)构建成二维在线联用系统。利用吉非替尼验证该系统的适用性,分子模拟对接实验研究活性组分与受体的相互作用方式,并利用MTT实验研究所筛选组分对EGFR细胞的体外抑制作用。结果表明,利用该系统可从射干总提取物中筛选出能够作用于EGFR的活性成分,并鉴定为次野鸢尾黄素;次野鸢尾黄素与EGFR的相互作用方式与阳性药吉非替尼相似;在浓度为0.4~50μmol/L范围内,吉非替尼、次野鸢尾黄素对高表达EGFR细胞的体外抑制作用具有剂量依赖性。EGFR细胞膜色谱在线HPLC-IT-TOF MS法有望成为从中草药中发现潜在抗肿瘤候选药物的有效方法。     

A statistical model for material removal prediction in polishing

This paper develops analytically a statistical model for predicting the material removal in mechanical polishing of material surfaces (MS). The model was based on the statistical theory and the abrasive–MS contact mechanisms. The pad-MS and pad-abrasive-MS interactions in polishing were characterised by contact mechanics. Two types of active abrasive particles in the polishing system were considered, i.e., Type I – the particles that can slide and rotate between the pad and MS, and Type II – those embedded in the pad without a rigid body motion. Accordingly, the material removal is considered to be the sum of the contributions from the two types of abrasive interactions. It was found that the mechanical properties and microstructure of the polishing pad and polishing conditions have a significant effect on the material removal rate, such as the porosity and elastic modulus of the pad, polishing pressure, volume concentration of abrasives, particle size, pad asperity radius and pad roughness. It was also found that different types of active particles contribute quite differently to the material removal. When the mean particle radius is small, the material removal is mainly due to the Type II particles, but when the mean particle radius becomes large, the Type I particles remove more materials. The model predictions are well aligned with experimental results available in the literature and can be used for the material removal prediction in chemo-mechanical polishing if a proper treatment of the chemical effect is introduced.     

A new technical approach to quantify cell-cell adhesion forces by AFM

Cell-cell adhesion is a complex process that is involved in the tethering of cells, cell-cell communication, tissue formation, cell migration and the development and metastasis of tumors. Given the heterogeneous and complex nature of cell surfaces it has previously proved difficult to characterize individual cell-cell adhesion events. Force spectroscopy, using an atomic force microscope, is capable of resolving such individual cell-cell binding events, but has previously been limited in its application due to insufficient effective pulling distances. Extended pulling range is critical in studying cell-cell interactions due to the potential for large cell deformations. Here we describe an approach to such experiments, where the sample stage can be moved 100 microm in the z-direction, by closed loop, linearized piezo elements. Such an approach enables an increase in pulling distance sufficient for the observation of long-distance cell-unbinding events without reducing the imaging capabilities of the atomic force microscope. The atomic force microscope head and the piezo-driven sample stage are installed on an inverted optical microscope fitted with a piezo-driven objective, to allow the monitoring of cell morphology by conventional light microscopy, concomitant with force spectroscopy measurements. We have used the example of the WM115 melanoma cell line binding to human umbilical vein endothelial cells to demonstrate the capabilities of this system and the necessity for such an extended pulling range when quantifying cell-cell adhesion events.     

Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM

A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research.     

Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells

The application of the conventional double-fixation method (glutaraldehyde and osmium tetroxide) to whole yeast cells is difficult because the thick cell wall of the yeast prevents the penetration of osmium tetroxide. However, this problem was solved by using the freeze-substitution fixation method. Therefore, it was possible to examine the intracellular structures of the yeast cells without digestion of the cell wall. In the present method, specimens for transmission electron microscopy and for scanning electron microscopy were prepared simultaneously. By scanning electron microscopic observation, three-dimensional information about internal structures was obtained. In the cytological analysis of the yeasts, intracellular structures were well preserved by using the freeze-substitution fixation method. On the outer leaflet of the nuclear envelope, many ribosomes were attached. The rough endoplasmic reticulum and Golgi apparatus were clearly seen in the yeast cytoplasm. The Golgi stack appeared to consist of smooth membranes, and small vesicles were present beside it. The details of other structures such as the nuclear division apparatus, actinlike filaments, and viruslike particles were also revealed. The present technique can be applied to most species of yeast cells. With this new information, the previous model of a yeast cell was modified.     

Rapid FlAsH labelling in the budding yeast Saccharomyces cerevisiae

Live cell imaging of protein distributions is an essential tool in modern cell biology. It relies on the functional labelling of a host protein with a fluorophore, which may either be a genetically fused fluorescent protein or an organic dye binding to the host protein. The biarsenical-tetracysteine system or 'FlAsH-labelling', is based on the high affinity interaction between a biarsenical probe and a small protein tag. This approach has been successfully used for live cell imaging in the budding yeast Saccharomyces cerevisiae. However, the established labelling protocols require a lengthy overnight incubation of the cells with the dye under tightly controlled growth conditions, which severely limits the use of this approach. In this study, we characterize an efficient method for introducing FlAsH-EDT(2) into live budding yeast cells using standard electroporation. The labelling time is reduced from more than 12 h to less than 1 h without compromising the labelling efficiency or cell viability. This approach may be used for cells in different growth phases or grown under different conditions. It may be further extended to other small high affinity probes, thus opening up new possibilities for labelling in budding yeast.     

碰撞诱导解离和高能碰撞解离方式进行蛋白质组学分析的比较

为了比较碰撞诱导解离（collision induced dissociation,CID）和高能碰撞解离（high energy collision dissociation,HCD）两种离子裂解模式在蛋白质组学分析方面的差异,分别应用这两种模式对牛血清白蛋白和细胞裂解物进行分析。通过比较所鉴定到的多肽和蛋白数目,发现在牛血清白蛋白中,HCD碎裂方法所得的谱图匹配率和多肽Mascot得分均较高,表明其质谱图质量较好。在复杂样品细胞裂解物分析中,CID碎裂方法鉴定到的谱图数、多肽数和蛋白数目均较多,表明该模式的灵敏度较高;HCD碎裂方法所得的谱图匹配率和Mascot得分均较高,表明其质谱图质量较好。因此,这两种模式均可用于大规模蛋白质组学分析,CID模式可提供更高的灵敏度,而HCD模式则可获得更高质量的质谱谱图。     

Design and implementation of RELab system to study the solar and wind energy

It is well known that renewable energy, today more than ever, has undergone an exponential development. Thus, it is necessary to create an efficient, compact and relatively cheap system to be used in education and research, which allows studying these types of energy. The concept, design and implementation of such a system – RELab are presented in this paper. The RELab system is a very useful tool for students and engineers to learn about the two types of renewable energy: solar and wind energy. The system has two components: the NI ELVIS platform and the RELab add-on. The RELab add-on is a modular one and it can be easily redesigned for using the photovoltaic cell, the wind turbine and the solar thermal collector. The software used is built in the graphical programming language LabVIEW, and it is flexible and open.