Set-up and application of a novel on-line nanoLC-CE-MS platform
新型在线nanoLC-CE-MS平台的建立和应用
基本信息
- 批准号:465662382
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:德国
- 项目类别:Research Grants
- 财政年份:
- 资助国家:德国
- 起止时间:
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
Proteins and their Post-translational modifications (PTMs) play a major role in diagnosis and potential treatment of various diseases. Du to low concentration and variability of these PTMs the direct characterization is still a major challenge in bioanalysis.In this proposed project we aim at developing a novel two-dimensional nanoliquid chromatography-capillary electrophoresis separation coupled to mass spectrometry (nanoLC-CE-MS) and apply this method initially for the analysis of intact (phosphorylated) proteins in biological samples. Such a system enables the combination of the advantages of both separation techniques: Reversed Phase chromatography allows for a preconcentration and an efficient separation of different proteins. Capillary electrophoresis enables the efficient separation of proteoforms especially when differing in charge (such as phosphorylation).Applying an insulated valve with integrated loops in the mid-nanoliter-range which we developed for CE-CE-MS, reversed phase nanoLC will be coupled to CE. At first, the set-up of such a nanoLC-CE coupling will be realized and important parameters for a successful coupling of these microfluidic techniques will be determined. Among others solvent compatibilities of the two separation systems, transfer volumes, and capillary coatings will be studied and optimized in order to obtain the most efficient separation in both dimensions. Furthermore, multiple heart-cut from the nanoLC and multi-segment injections (injection of samples while previous separation still takes place) into the CE dimension will be evaluated for most efficient separation.For unequivocal identification and localization of the PTMs the nanoLC-CE separation will be coupled via a nanoelectrospray interface to top-down MS/MS analysis. This set-up will be applied for identification and characterization of proteoforms of intact proteins in biological samples in both targeted and untargeted approaches.In a subsequent funding period we plan to include both various other chromatographic separations (such as ion chromatography or hydrophilic liquid interaction chromatography) and a comprehensive LC-CE coupling. Furthermore, applications to clinical questions will be implemented.
蛋白质及其翻译后修饰(PTMS)在诊断和潜在治疗各种疾病中起主要作用。这些PTM的DU到低浓度和可变性,直接表征仍然是生物分析中的主要挑战。在这项拟议的项目中,我们旨在开发一种新型的二维纳米液体色谱 - 含量电泳分离耦合到质谱法(Nanolc-CE-MS)(Nanolc-CE-MS)(Nanolc-CE-MS)(最初)进行生物分析(量基)(量量)。这样的系统可以结合两种分离技术的优势:相反的相色谱法允许对不同蛋白质进行预浓缩和有效的分离。毛细血管电泳可以有效地分离蛋白质成型,尤其是当电荷不同时(例如磷酸化)。在我们为CE-CE-MS开发的中纳溶剂范围内,将绝缘阀与综合循环播放,将其相反的相位nanolc耦合到CE。首先,将实现这种纳米ce耦合的设置,并确定成功的参数以成功地耦合这些微流体技术。将研究和优化两个分离系统的溶剂兼容性,转移量和毛细血管涂层,以在两个维度中获得最有效的分离。此外,将评估来自纳米克和多段注射的多个心脏(在先前的分离时注射样品,在仍然进行的样品中注入)将评估到CE维度中,以进行最有效的分离。对于PTMS的最高识别和定位,Nanolc-CE分离将通过nanolelelelectRosPray interface intopsface topsface tops-ms mss/ms MSS s MSS-MS MSS-MS MSS MSS-MS MSS MSS-MS MS MS MS MS MS MS MS MS MSS-MS MSS MSS-MS MS MS MSS-MS MSS MSS-MS MSS DY-MSS-MS MSS MSS-MS MSS-MS MSS-MS MSS-MS MSS-MS MSS-MS MSS-MS M.该设置将用于在靶向和无靶方法的生物样品中鉴定和表征完整蛋白质的蛋白质成型。此外,将实施对临床问题的申请。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Professor Dr. Christian Neusüß其他文献
Professor Dr. Christian Neusüß的其他文献
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{{ truncateString('Professor Dr. Christian Neusüß', 18)}}的其他基金
Novel Successive Multi-Ionic Layers for ultra-efficient, diffusion-limited separation of intact proteins by capillary electrophoresis and capillary electrophoresis-mass spectrometry
新型成功的多离子层,可通过毛细管电泳和毛细管电泳-质谱法对完整蛋白质进行超高效、扩散限制的分离
- 批准号:
446330632 - 财政年份:
- 资助金额:
-- - 项目类别:
Research Grants
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