Sensitive targeted multiple protein quantification based on elemental detection of Quantum Dots

A generic strategy based on the use of CdSe/ZnS Quantum Dots (QDs) as elemental labels for protein quantification, using immunoassays with elemental mass spectrometry (ICP-MS), detection is presented. In this strategy, streptavidin modified QDs (QDs-SA) are bioconjugated to a biotinylated secondary antibody (b-Ab₂). After a multi-technique characterization of the synthesized generic platform (QDs-SA-b-Ab₂) it was applied to the sequential quantification of five proteins (transferrin, complement C3, apolipoprotein A1, transthyretin and apolipoprotein A4) at different concentration levels in human serum samples. It is shown how this generic strategy does only require the appropriate unlabeled primary antibody for each protein to be detected. Therefore, it introduces a way out to the need for the cumbersome and specific bioconjugation of the QDs to the corresponding specific recognition antibody for every target analyte (protein). Results obtained were validated with those obtained using UV–vis spectrophotometry and commercial ELISA Kits.     

关于带宽极值问题的一些结果

本文研究的问题是确定f(p,B）的值，也就是给定顶点数p和带宽B，求满足最大度不超过B的连通图的最小边数，本文给出了一些f(p,B）的值及相应极图。     

Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.     

Comparison of copper labeling followed by liquid chromatography-inductively coupled plasma mass spectrometry and immunochemical assays for serum hepcidin-25 determination

Hepcidin-25 has been defined as the key biomarker in iron metabolism. This peptide binds to the iron transporter ferroportin to cause its degradation. Therefore, the need for specific, accurate and precise methods for the quantification of hepcidin-25 in biological fluids is dramatically increasing. In this regard, the use of rapid immunochemical methods that provide low limit of quantification is desired for routine clinical use. However, such fast methodologies should be first analytically evaluated and compared with alternative strategies to check for their advantages and limitations. Here we compare the use of a commercial immunochemical assay for hepcidin determination with a novel analytical approach based on Cu-labeling of the peptide followed by Cu determination using liquid chromatography (HPLC) and plasma mass spectrometry (ICP-MS). The figures of merit of both systems reveal similar analytical characteristics and both seem to be adequate for the determination of the peptide at biologically relevant concentrations in human serum samples. The analysis of a larger number of samples (n = 50) by both techniques showed a good agreement in the concentrations found. Such finding permits to address the hepcidin recovery in the sample preparation procedure necessary for the HPLC-ICP-MS analysis in human serum that turn out to be 76–85%. Additionally, limitations due to cross-reactivity issues of the ELISA method could be addressed in some of the samples by using LC-ICP-MS and were confirmed by LC-Electrospray-MS.     

Comparison of oligosaccharide labeling employing reductive amination and hydrazone formation chemistries

In this work, we compare labeling by two negatively charged fluorescent labels, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and 8-(2-hydrazino-2-oxoethoxy)pyrene-1,3,6-trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4-hydroxybenzaldehyde and maltoheptaose followed by LC/UV-MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV-MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N-linked glycan analysis by CE/LIF.     

小分子荧光探针在蛋白质标记与成像分析中的应用

小分子荧光探针由于其体积小、合成简单等特点在蛋白质成像技术中扮演着越来越重要的角色。此领域的研究融合了生物化学、有机合成、分析化学等相关学科,是当今化学发展的一个重要方向,有着广阔的前景。目前,能够专一性地与目标蛋白质(POI)结合的小分子探针较少,设计和合成方法的缺乏已经成为制约该领域进一步发展的瓶颈。本文概括地介绍了近年来出现的一些小分子荧光探针,关注它们在活体标记中的应用。     

211At标记蛋白质或多肽的方法研究

α核素211At具有良好的辐射生物学性质,以对肿瘤细胞具有高亲和性的单克隆抗体或多肽类配体为载体则是实现211At肿瘤靶向治疗的最理想方式之一。本文介绍了211At标记蛋白质或多肽的方法的现状与进展,对存在的一些问题及今后的发展方向进行了讨论。     

7-甲氧基香豆素-3-羧酸-N-琥珀酰亚胺酯用于神经肽的标记与分析

选用7-甲氧基香豆素-3-羧基-N-琥珀酰亚胺酯(MCSE)作为衍生试剂, 并借助高效液相色谱和质谱等仪器对甲硫脑啡肽、亮脑啡肽和神经紧张素等3种神经肽进行了标记与分析.     

SERS标记纳米粒子用于免疫识别

激光拉曼光谱技术近年来已成为研究生物分子结构常用的光谱手段.尤其在研究水溶液中蛋白质的结构和构象方面发挥了重要作用.然而,常规拉曼光谱的信号强度很低,限制了其在各个领域中的应用.表面增强拉曼光谱(SERS)和表面增强共振拉曼光谱(SERRS)技术可使信号增强6～10个数量级,尤其是SERS技术已发展到检测单分子的水平,更为其在生物方面的应用开拓了新的局.