Application of plug–plug technique to ACE experiments for discovery of peptides binding to a larger target protein: A model study of calmodulin‐binding fragments selected from a digested mixture of reduced BSA

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a “plug–plug” technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan‐X, β‐endorphin, and oxytocin, as candidates for calmodulin‐binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug–plug technique, the ACE–MS screening methodology successfully selected calmodulin‐binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with K_d values between 8–147 μM for calmodulin were obtained from a Glu‐C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE–MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.     

Quality by design: A systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column

The present work describes the systematic development of a robust, precise, and rapid reversed‐phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality‐by‐design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters—column temperature, gradient time, and flow rate using eight experiments—to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused‐core C₁₈ (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry.     

Development of an automated dual‐mode supercritical fluid chromatography and reversed‐phase liquid chromatography mass‐directed purification system for small‐molecule drug discovery

We report the development of a dual‐mode mass‐directed supercritical fluid chromatography and reversed‐phase liquid chromatography purification system. The addition of a third pump allows for flexible mobile phase control between the two techniques, and enables operation of either chromatography mode within minutes by activation of a set of switching valves on a single system. Software control, fluidic pathways, interface to the mass spectrometer, and fraction collection have been modified for compatibility between both separation methods. The conditioning solvent and tuning parameters for the mass spectrometer were adjusted to achieve an ideal signal trace in either mode with good linearity (r² > 0.970) over a range of concentrations and minimal noise for accurate peak detection and isolation. The registration success rate is 90% and overall sample recovery for either technique is 80?90%. Combining two orthogonal separation and purification modes in one single system has improved the purification throughput of complex mixtures and has been a valuable, cost‐saving tool in our laboratory.     

Urinary metabolomic study of non‐small cell lung carcinoma based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Metabolic profiles from human urine reveal the significant difference of carnitine and acylcarnitines levels between non‐small cell lung carcinoma patients and healthy controls. Urine samples from cancer patients and healthy individuals were assayed in this metabolomic study using ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. The data were normalized by the sum of all intensities and creatinine calibration, respectively, before orthogonal partial least squares discriminant analysis. Twenty differential metabolites were identified based on standard compounds or tandem mass spectrometry fragments. Among them, some medium‐/long‐chain acylcarnitines, for example, cis‐3,4‐methylene heptanoylcarnitine, were found to be downregulated while carnitine was upregulated in urine samples from the cancer group compared to the control group. Receiver operating characteristic analysis of the two groups showed that the area under curve for the combination of carnitine and 11 selected acylcarnitines was 0.958. This study suggests that the developed carnitine and acylcarnitines profiling method has the potential to be used for screening non‐small cell lung carcinoma.     

Urine sample preparation for proteomic analysis

Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross‐reactions that should be taken into consideration. The incorporation of new post‐translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery.     

Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics†

In current shotgun‐proteomics‐based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.     

On‐line solid‐phase extraction for liquid chromatography–mass spectrometry analysis of pesticides

Public concern about pesticides in food and water has increased dramatically in the last two decades. In order to guarantee consumers’ health and safety, analytical methods that could provide fast and reliable answers without compromising accuracy and precision are required. Sample treatment is probably the most tedious and time‐consuming step in many analytical procedures and, despite the significant advances in chromatographic separations and mass spectrometry techniques, sample treatment is still one of the most important parts of the analytical process for achieving good analytical results. Therefore, over the last years, considerable efforts have been made to simplify the stage and to develop fast, accurate, and robust methods that allow the determination of a wide range of pesticides without compromising the integrity of the extraction process. This review article intends to give a short overview of recently developed on‐line solid‐phase extraction, preconcentration, and clean‐up procedures for the determination of pesticides in complex matrices by liquid chromatography–mass spectrometry techniques.     

异原子配位结构碳基单原子电催化剂结构与性能相关性的研究进展

单原子催化剂是一类以相互孤立的单个金属原子作为催化活性中心的、 具有高原子经济性及高活性的负载型催化剂, 被广泛应用于能源电催化领域. 近年来, 通过使用两种或两种以上原子与活性中心金属原子配位, 构建具有异原子配位结构的单原子材料, 展现了优异的电催化性能. 研究发现, 这种不对称的配位结构有效调控了中心金属原子的电子结构, 优化了催化反应的吸附和脱附能量, 提高了电催化的性能. 本文综合评述了具有异原子配位结构碳基单原子电催化剂的合成策略、 表征技术与方法, 以及在前沿能源电催化应用中的催化剂性能与结构之间的构效关系, 并展望了异原子配位结构碳基单原子电催化剂的研究前景.     

冰冻人血清中尿酸标准物质复制的定值EI北大核心CSCD

为了满足该标准物质的需求,进行了第二次尿酸血清标准物质的复制。针对复制的尿酸血清标准物质,基于单四极杆质谱的液相色谱-同位素稀释质谱法(LC-IDMS),用乙腈沉淀法去除蛋白质,BEH C18色谱柱和电喷雾离子源负离子模式(ESI^(-)),同位素稀释的单点校准法进行定值、均匀性检验、稳定性检验以及不确定度评定等研究。此定值方法经过CCQM-K109(血清中尿酸分析)国际关键比对进行验证。复制的2种不同浓度水平尿酸(肾病患者和正常人)血清标准物质的定值结果分别为(73.5±1.3)μg/g,(47.5±1.1)μg/g,其均匀性和稳定性评估结果良好。     

Recent Progress in Mass Spectrometry-based Metabolomics for Colorectal Cancer

Metabolomics, one of the latest omics technologies, is employed to reveal overall metabolic trajectories, identify disease causative mechanisms and provide information for preventive diagnosis and drug targeting. Cancer is a disease known to alter cellular metabolism and so metabolomics can play an important role in the early diagnosis of cancer and in the evaluation of medical interventions and treatments for cancer. Many metabolomics studies rely on high-sensitive and high-throughput mass spectrometry platforms. In recent years, various mass spectrometry(MS) methodologies have been developed and enriched the scope of metabolite detection, contributing to disease studies, such as diabetes, cancer, and depression. Colorectal cancer is the third most diagnosed cancer worldwide and its incidence ranked third in China. This review focuses on the mass spectrometry technologies in metabolomics and summarizes the progress of metabolomics research in colorectal cancer.