基于电感耦合等离子体质谱的蛋白质定量技术研究进展

综述了ICP-MS法应用于蛋白质定量技术方面的研究进展.蛋白质定量研究已成为蛋白质组学研究领域的热点,它是解析生物体蛋白质功能的重要途径.基于同位素标记和生物质谱分析技术是蛋白质定量最常用的方法之一,近年来,随着质谱技术的发展,电感耦合等离子体质谱(ICP-MS)技术成为元素测量的重要手段,这使其在蛋白质定量中具一定的应用前景.     

原子荧光和电感耦合等离子体质谱法在硒形态分析中的应用

硒是人体必需的微量元素之一,具有抗肿瘤、防衰老、防辐射和增强机体免疫力等多种功能。近年来,随着人们生活水平的不断提高,各种各样的富硒产品及富硒保健品走进了我们的生活,硒的形态测定越来越受到人们广泛的关注。本文对目前主要检测硒形态的两种方法—原子荧光法(AFS)和电感耦合等离子质体谱法(ICP-MS)在食品、水果蔬菜、富硒保健品、生物样品等方面的应用进行了综述。     

基于流式细胞仪高通量分选的深海微生物单细胞培养

[目的]建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法,通过该方法从印度洋深海样品中分离微生物纯培养菌株.[方法]利用流式细胞仪单细胞分选功能,以前向角(FSC)和侧向角(SSC)散射光信号代替荧光信号作为分选逻辑,对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养.[结果]确定了流式细胞分选的区域和...     

神经系统基因表达的单细胞分析

神经系统基因表达的单细胞分析宋萍包永德（中国科学院上海生理研究所,上海２０００３１）关键词单细胞分析ＲＴ－ＰＣＲ神经元某种神经元区别于非神经元以及其它类型的神经细胞的各种性质反映出在基因表达方式上必然存在着某些特异性的差别［１］。现代神经生物学中亟待...     

重庆市野生黄精中重金属含量分析与评价

目的：建立电感耦合等离子体质谱法（ICP-MS）测定不同产地黄精中砷、铅、铬、镉、汞含量的方法,进行含量分析,为黄精的医用和食疗安全提供理论基础。方法：采用ICP-MS法对52份野生黄精样品中5种重金属进行检测,内标法定量,并进行数据分析。结果：野生黄精中5种重金属的方法检出限为0.002～0.020 mg/kg,回收率为95.5%～104.4%,精密度为2.8%～4.6%;野生黄精根茎中含量高低依次为铬、铅、镉、砷和汞,各元素间呈正相关;巫山的铬和砷显著高于其他地区,奉节的铅最高,石柱的镉显著高于其他地区;黄精不同部位的重金属含量高低：根须>根茎>叶子>种子,其中根须的铬含量高达4.406±1.021 mg/kg。结论：野生黄精的重金属含量存在一定的地域性差异;不同部位的含量也有差异：地下部位（根须、根茎）含量大于地上部位（叶子、种子）。本研究可为黄精的外源性污染提供数据支撑。     

小花清风藤中金属元素的ICP-MS分析

分析了采自中国广西清风藤科植物小花清风藤中13种金属元素的含量。小花清风藤茎叶样品经微波消解处理,采用电感耦合等离子体质谱（inductively coupled plasma massspectrometry,ICP-MS）技术检测。钛、钒、铬、锰、铁、铜、锌、砷、硒、铅10种元素采用普通模式检测;为消除多原子离子的干扰,铝、钴和镍3种元素采用了碰撞/反应池技术（collision/reaction cell technology）检测。结果表明试验条件下,硒元素未检出,各金属元素的方法检出限（3s）在0.083~1.564 μg/L之间,方法精密度（n=8）在0.028%~1.510%之间,加标回收率在90%~110%之间。研究结果为了解小花清风藤中金属元素的种类与含量,进一步开发利用小花清风藤提供了参考。     

ICP-MS法测定不同基质保健品中10种微量元素的方法

目的：采用微波消解—电感耦合等离子质谱法（ICP-MS）分别对液体水基、固体颗粒基、固体片剂和软胶囊4种不同基质的保健品中的钾、钙、钠、镁、铁、锰、铜、锌、铅、砷进行同时分析测定。方法：样品经过微波消解后,使用电感耦合等离子质谱仪分别对4种基质的10种元素进行同时测定。结果：经测定,该方法的线性范围宽,线性良好,线性相关系数均在0.999以上;灵敏度高,各元素的检出限在0.008~3.9mg/kg之间;10种元素的回收率在85.0%~109%之间,精密度在1.2%~9.7%之间。结论：本方法能方便、准确、灵敏的对液体水基、固体颗粒基、固体片剂和软胶囊4种不同基质的保健品种10种元素进行同时测定。     

ICP-MS法测定茶多酚片中铅和总砷的不确定度分析

目的：为提高电感耦合等离子质谱法（ICP-MS）测定茶多酚片中痕量重金属铅和总砷含量的准确性,通过建立数学模型,对不确定度来源进行分析。方法：样品经过微波消解前处理,采用ICP-MS法分别测定茶多酚片中铅和总砷的含量,根据JJF1059.1-2012《测量不确定度评定与表示》分析不确定度来源,计算合成不确定度和扩展不确定度。结果：经过分析计算,茶多酚片中铅和总砷的扩展不确定度分别为0.029、0.010mg/kg。结论：影响茶多酚片中铅和总砷ICP-MS法测定的不确定度主要来源为样品前处理和标准溶液。     

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.¹ The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 μm wavelength utilize the sudden excitation of water that results in phase explosion.² Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.^3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.¹² This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.     

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.¹ Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.^2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.^6-7Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.     

Novel Bioimaging Techniques of Metals by Laser Ablation Inductively Coupled Plasma Mass Spectrometry for Diagnosis Of Fibrotic and Cirrhotic Liver Disorders

Background and Aims

Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult.

Methods

In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers.

Results

Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples.

Conclusions

Biometal imaging via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.     

单细胞RNA测序数据分析方法研究进展

单细胞RNA测序(Single cell RNA sequencing,scRNA-Seq)已经广泛应用于细胞分化、肿瘤微环境及多种疾病病因学研究。目前,由于scRNA-Seq具有低捕获率、高噪声、高变异性等特点,通过优化数据分析方法提高测序结果准确性已经成为测序领域的研究热点。对近年来数据分析过程中利用的数学方法进行了总结,讨论了数据分析的优势及存在的问题,以期为新算法的开发和应用提供参考,逐步提高测序结果的可靠性。     

Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry

1. Download high-res image (196KB)
2. Download full-size image

     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Translating Cancer Molecular Variability into Personalized Information Using Bulk and Single Cell Approaches

Cancer research is striving toward new frontiers of assigning the correct personalized drug(s) to a given patient. However, extensive tumor heterogeneity poses a major obstacle. Tumors of the same type often respond differently to therapy, due to patient‐specific molecular aberrations and/or untargeted tumor subpopulations. It is frequently not possible to determine a priori which patients will respond to a certain therapy or how an efficient patient‐specific combined therapy should be designed. Large‐scale datasets have been growing at an accelerated pace and various technologies and analytical tools for single cell and bulk level analyses are being developed to extract significant individualized signals from such heterogeneous data. However, personalized therapies that dramatically alter the course of the disease remain scarce, and most tumors still respond poorly to medical care. In this review, the basic concepts of bulk and single cell approaches are discussed, as well as their emerging role in individualized designs of drug therapies, including the advantages and limitations of their applications in personalized medicine.     

Front Cover: A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution (J. Biophotonics 2/2018)

A new type of high‐throughput imaging flow cytometer (>20 000 cells s^‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )