Biophysics Reports

2015 Vol. 1, No. 2

Cover Story

A convenient and highly ef icient magnetic solution that can selectively enrich saccharides in one step for glycomic analysis was described. In this approach, 2-(2-aminoethoxy) ethanol blocked phenylboronic acid (PBA) functionalized magnetic nanoparticles (blocked PMNPs) were fabricated, and employed to make targets magnetic, so that the blocked PMNPs bound targeted molecules can be easily separated from the solution by applying an assistant magnetic ield. The PBA groups can speci ically recognize the cis-diols moieties of saccharides, forming a reversible covalent boronate ester bond. The blocking molecules, 2-(2-aminoethoxy) ethanol, enable to deplete the nonspeci ic absorption of the undesirable interferent that always co-exists with saccharides. Finally, the enriched saccharides bound on PMNPs can be directly analyzed by MALDI-TOF MS without extra steps to separate them from the PMNPs. The combination of magnetic separation, selective enrichment, and sensitive detection make our PMNPs-MALDI-TOF approach a highly ef icient, sensitive and high-throughput tool for saccharides analysis.

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Phenylboronic acid-functionalized magnetic nanoparticles for one-step saccharides enrichment and mass spectrometry analysis

Xiangdong Xue, Yuanyuan Zhao, Xu Zhang, Chunqiu Zhang, Anil Kumar, Xiaoning Zhang, Guozhang Zou, Paul C. Wang, Jinchao Zhang, Xing-Jie Liang

2015, 1(2): 61-70. doi: 10.1007/s41048-015-0002-3

Abstract(107)

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Abstract:
In this work, 2-(2-aminoethoxy) ethanolblocked phenylboronic acid-functionalized magnetic nanoparticles (blocked PMNPs) were fabricated for selective enrichment of different types of saccharides. The phenylboronic acid was designed for capturing the cis-diols moieties on saccharides molecules, and the 2-(2-aminoethoxy) ethanol can deplete the nonspecific absorption of peptides and proteins which always coexisted with saccharides. For mass spectrometry analysis, the PMNPs bound saccharides can be directly applied onto the MALDI plate with matrix without removing the PMNPs. By PMNPs, the simple saccharide (glucose) could be detected in pmol level. The complex saccharides can also be reliably purified and analyzed; 16 different Nglycans were successfully identified fromovalbumin, and the high-abundance N-glycans can be detected even when the ovalbumin was in very low concentration (2 μg). In human milk, ten different oligosaccharides were identified, and the lactose can still be detected when the human milk concentration was low to 0.01 μL.

A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation

Dongwan Cheng, Li Zheng, Junjie Hou, Jifeng Wang, Peng Xue, Fuquan Yang, Tao Xu

2015, 1(2): 71-80. doi: 10.1007/s41048-015-0012-1

Abstract(321)

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Abstract:
The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.

A comprehensive procedure for antiviral inhibitor discovery using EV71 as an example

Lin Cao, Shouhai Zhu, Yaxin Wang, Zhiyong Lou, Yuna Sun

2015, 1(2): 81-89. doi: 10.1007/s41048-015-0006-z

Abstract(186)

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Abstract:
The prevalence of chronic viral infectious diseases, the emergence and re-emergence of new viral infections, and in particular, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. Increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. In this work, we introduce a comprehensive system using enteroviruses 71 (EV71) as an example for leading compound discovery to develop new antiviral.

Skeletal intramyocellular lipid metabolism and insulin resistance

Yiran Li, Shimeng Xu, Xuelin Zhang, Zongchun Yi, Simon Cichello

2015, 1(2): 90-98. doi: 10.1007/s41048-015-0013-0

Abstract(244)

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Abstract:
Lipids stored in skeletal muscle cells are known as intramyocellular lipid (IMCL). Disorders involving IMCL and its causative factor, circulatory free fatty acids (FFAs), induce a toxic state and ultimately result in insulin resistance (IR) in muscle tissue. On the other hand, intramuscular triglyceride (IMTG), the most abundant component of IMCL and an essential energy source for active skeletal muscle, is different from other IMCLs, as it is stored in lipid droplets and plays a pivotal role in skeletal muscle energy homeostasis. This review discusses the association of FFA-induced ectopic lipid accumulation and IR, with specific emphasis on the relationship between IMCL/IMTG metabolism and IR.

The application of traditional transmission electron microscopy for autophagy research in Caenorhabditis elegans

Attila L. Kovács

2015, 1(2): 99-105. doi: 10.1007/s41048-015-0014-z

Abstract(295)

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Abstract:
Traditional ultrastructural characterization of autophagic processes remains an important approach to be used in parallel with molecular genetics, light microscopy, and other methods. The special nature of Caenorhabditis elegans as an object for transmission electron microscopy makes its introduction into autophagy research a challenging task. The basis of the protocol to prepare C. elegans samples for autophagy studies was worked out around the turn of the millennium and has been used since then in my laboratory with some modifications. The method described here enables the user to prepare samples for systematic morphologic as well as morphometric investigations to characterize autophagy with a high but still realistic investment of effort.

Transcriptomic analysis of human breast cancer cells reveals differentially expressed genes and related cellular functions and pathways in response to gold nanorods

Teng Zhou, Yipeng Du, Taotao Wei

2015, 1(2): 106-114. doi: 10.1007/s41048-015-0005-0

Abstract(105)

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Abstract:
Breast cancer is the leading cause of cancer deaths inwomen.Recent advances in nanomedicine have shown that gold nanorods (AuNRs), as multifunctional drug delivery and photothermal therapeutic agents, have potential for use in cancer therapy. However, the effect of AuNRs on the transcriptome of breast cancer cells is unknown. In the present study, cells of the triple-negative human breast cancer cell line MDA-MB-231, which has high metastatic activity, were treated with AuNRs for transcriptomic analysis using RNA-seq technology. In total, 3126 genes were found to be up-regulated and 3558 genes were found to be downregulated inAuNR-treatedMDA-MB-231 cells. These differentially expressed genes presumably take part in multiple biological pathways, including glycolysis and regulation of the actin cytoskeleton, and impact a variety of cellular functions, including chemoattractant activity. The distinct gene expression profile of MDA-MB-231 cells treated with AuNRs provides a foundation for further screening and validation of important genes involved in the interaction between AuNRs and MDA-MB-231 cells.