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.     

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     

Synthesis and Thermal Performance of Polyurea Microcapsulated Phase Change Materials by Interfacial Polymerization

Microencapsulated phase change materials with paraffin as the core material were synthesized by interfacial polymerization of isophorone diisocyanate with diethylene triamine. The particle size and particle size distribution, morphology, thermal performance and the encapsulation efficiency of the prepared materials were investigated. The results of Fourier transform infrared spectrometer and X-ray photoelectron spectroscopy suggested that the paraffin core was well encapsulated by the polyurea resin. The particle size of the prepared materials decreased and its distribution became narrow with the increase of the emulsification time, stirring speed and emulsifier amount. The thermal gravimetric analysis indicated that the prepared materials exhibited good thermal stability, while the differential scanning calorimetry their high encapsulation efficiency.     

Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein

Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexisting cis- and trans-isomers, holds significance as an archetypal system for LSS emission due to excited-state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time-resolved electronic spectroscopies, aided by quantum calculations, to dissect the cis- and trans-mKeima photocycle from ESPT, isomerization, to ground-state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonated trans-isomer governs LSS emission at 620 nm, while the deprotonated cis-isomer's 520 nm emission is weak due to an ultrafast cis-to-trans isomerization. Complementary spectroscopic techniques as a table-top toolset are thus essential to study photochemistry in physiological environments.