LC–MS bioanalysis of intact proteins and peptides

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC–MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC–MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC–MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated. For peptides, topics including quantitation of intact peptide vs. digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard and sample preparation are discussed.     

介质材料二次电子发射特性对微波击穿的影响

以介质填充的平行板放电结构为例,本文主要研究了介质填充后微波低气压放电和微放电的物理过程.为了探究介质材料特性对微波低气压放电和微放电阈值的影响,本文采用自主研发的二次电子发射特性测量装置,测量了7种常见介质材料的二次电子发射系数和二次电子能谱.依据二次电子发射过程中介质表面正带电的稳定条件,计算了介质材料稳态表面电位与二次电子发射系数以及能谱参数的关系.在放电结构中引入与表面电位相应的等效直流电场后,依据电子扩散模型和微放电中电子谐振条件,分别探讨了介质表面稳态表面电位的大小对微波低气压放电和微放电阈值的影响.结果表明,介质材料的二次电子发射系数以及能谱参数越大,介质材料的稳态表面电位也越大,对应的微波低气压放电和微放电阈值也越大.所得结论对于填充介质的选择有一定的理论指导价值.     

Nanosized Carbon Macrocycles Based on a Planar Chiral Pseudo Meta-[2.2]Paracyclophane

Structural designs combining cycloparaphenylenes (CPPs) backbone with planar chiral [2.2]paracyclophane ([2.2]PCP) lead to optical-active chiral macrocycles with intriguing properties. X-ray crystal analysis revealed aesthetic necklace-shaped structures and size-dependent packages with long-range channels. The macrocycles exhibit unique photophysical properties with high fluorescence quantum yield of up to 82 %, and the fluorescent color varies with ring size. In addition, size-dependent chiroptical properties with moderately large CPL dissymmetry factor of 10⁻³ and CPL brightness in the range of 30–40 M⁻¹ cm⁻¹ were observed.     

Organometallic titanocene complex as highly efficient bifunctional catalyst for intramolecular Mannich reaction

Bifunctional catalysts bearing two catalytic sites, Lewis acidic organometallic titanocene and Brønsted acidic COOH, have been assembled in situ from Cp₂TiCl₂ with carboxylic acid ligands, showing high catalytic activity over an intramolecular Mannich reaction towards synthesis of 2‐aryl‐2,3‐dihydroquinolin‐4(1H)‐ones. The determination of the bifunctional catalyst Cp₂Ti(C₈H₄NO₆)₂ was elucidated by single X‐ray HR‐MS and investigation of catalytic behavior. In particular, masking the Brønsted acidic COOH catalytic site with dormant COOMe lowered the reaction yield greatly, indicating that two catalytic sites work together to maintain high catalytic efficiency.     

XANES/EPR Evidence of the Oxidation of Nickel(II) Quinolinylpropioamide and Its Application in Csp3−H Functionalization

An in situ generated oxidation species of nickel quinolinylpropioamide intermediate was produced. Characterization by X-ray absorption near edge structure (XANES) and EPR provides complementary insights into this oxidized nickel species. With aliphatic amides and isocyanides as substrates, a nickel-catalyzed facile synthesis of structurally diverse five-membered lactams could be achieved.     

Solubilization of Hexyl Aminolevulinate by Surfactants for Tumor Fluorescence Detection

Hexyl aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinic acid (5-ALA) and can induce more protoporphyrin IX (PpIX) formation and stronger fluorescence intensity (FI) than 5-ALA, which will greatly facilitate photodynamic diagnosis and therapy. The main drawback of HAL is its low solubility in neutral aqueous media. In this study, surfactants were used to increase HAL solubility in the cell culture medium and serum, followed by in vitro fluorescence formation measurement in human pancreatic cancer cells (SW1990) and in vivo fluorescence detection in tumor-bearing mice. The results showed that Tween 80 (TW80) and Kolliphor® HS 15 (HS15) increased the solubility of HAL in the selected media. Although TW80 and HS15 exhibited in vitro cytotoxicity at high concentrations (5 mg mL⁻¹), they facilitated fluorescent signal formation at the early stage of cell incubation. When surfactants were used, the FI should be determined without the routine washing process because surfactant-containing culture medium caused the loss of synthesized PpIX during the washing process. When HAL dissolved in TW80 solution was injected intraperitoneally into pancreatic cancer-bearing mice at a dose of 50 mg kg⁻¹, the tumors exhibited red fluorescence, which indicated that systemic administration of surfactant-solubilized HAL might be applicable for tumor fluorescence detection in vivo.     

Conjugated polymer nanoparticles with aggregation induced emission characteristics for intracellular Fe3+ sensing

In this article, a novel zwitterionic conjugated polyelectrolyte containing tetraphenylethene unit was synthesized via Pd‐catalyzed Sonogashira reaction. The resulting polymer (P2), which exhibited typical aggregation‐induced emission (AIE) properties, was weakly fluorescent in dilute DMSO solution and showed bright fluorescence emissions when aggregated in DMSO/water mixtures or fabricated into conjugated polymer nanoparticles (CPNs). The nanoparticles from P2 could be prepared by reprecipitation method with an average diameter around 23 nm. Notably, the cell‐staining efficiencies of lipid‐P2 nanoparticles could be enhanced with lipid encapsulation and these nanoparticles were endocytosed via caveolae‐mediated and clathrin‐mediated endocytosis pathways. Furthermore, the lipid‐P2 nanoparticles with low cytotoxicity, high photostability and efficient cell staining ability could be employed for in vitro detection of Fe³⁺ ions in A549 cells. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1686–1693