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Step-Wise Assessment and Optimization of Sample Handling Recovery Yield for Nanoproteomic Analysis of 1000 Mammalian Cells

机译:纳米蛋白分析的纳米蛋白分析样本处理回收产量的逐步评估和优化

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摘要

Protein and peptide adhesion is a major factor contributing to sample loss during proteomic sample preparation workflows. Sample loss often has detrimental effects on the quality of proteomic analysis by compromising protein identification and data reproducibility. When starting with a low sample amount, only the most abundant proteins can be identified, which often offers little insights for biological research. Although the general idea about severe sample loss from low amount of starting material is widely presumed in the proteomics field, quantitative assessment on the impact of sample loss has been poorly investigated. In the present study, we have quantitatively assessed sample loss during each step of a conventional in-solution sample preparation workflow using bicinchoninic acid (BCA) and targeted LC/MS/MS protein and peptide assays. According to our assessment, for starting materials of similar to 1000 mammalian cells, surface adhesion, along with desalting and speed-vacuum drying steps, all contribute heavily to sample loss, in particular for low-abundance proteins. With this knowledge, we have adapted slippery liquid infused porous surface (SLIPS) treatment, commercial LoBind tubes, and in-line desalting during sample processing. With these improvements, we were able to use a conventional in-solution sample handling method to identify on average 829 proteins with 1000 U2OS osteosarcoma cells (similar to 100 ng) with 75-min LC/MS/MS runs, an 11-fold increase in protein identification. Our optimized in-solution workflow is straightforward and also much less equipment-and technique-demanding than other advanced sample preparation protocols in the field.
机译:蛋白质和肽粘附是有助于在蛋白质组样品制备工作流期间进行样品损失的主要因素。样品损失通常对蛋白质鉴定和数据再现性损害蛋白质组学分析的质量具有不利影响。在以低样品量开始时,只能识别最丰富的蛋白质,这通常会对生物学研究提供很少的见解。虽然在蛋白质组学领域中广泛推出了对低量起始材料的严重样品损失的一般思路,但对样品损失的影响的定量评估已经较差。在本研究中,我们在使用双子胆酸(BCA)和靶向LC / MS / MS蛋白和肽测定的常规溶液样品制备工作流程的每个步骤期间定量评估了样品损失。根据我们的评估,对于类似于1000个哺乳动物细胞的原料,表面粘附以及脱盐和速度真空干燥步骤,所有这些都有助于样品损失,特别是对于低丰度蛋白。通过这种知识,我们在样品加工过程中,我们在样品加工过程中适应了滑湿的液体注入多孔表面(SLIPS)处理,商业LOBIND管和在线脱盐。通过这些改进,我们能够使用常规的溶液样品处理方法来识别平均829个蛋白质,其中1000μS骨肉瘤细胞(类似于100ng),具有75分钟的LC / MS / MS运行,增加11倍在蛋白质鉴定中。我们优化的内部工作流程是简单的,并且还比其他先进的样品制备方案更少的设备和技术要求。

著录项

  • 来源
    《Analytical chemistry》 |2019年第16期|共6页
  • 作者单位

    McMaster Univ Dept Biochem &

    Biomed Sci Hamilton ON L8S 4K1 Canada;

    McMaster Univ Dept Biochem &

    Biomed Sci Hamilton ON L8S 4K1 Canada;

    McMaster Univ Sch Biomed Engn Hamilton ON L8S 4K1 Canada;

    McMaster Univ Dept Mech Engn Hamilton ON L8S 4K1 Canada;

    McMaster Univ Dept Biochem &

    Biomed Sci Hamilton ON L8S 4K1 Canada;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分析化学;
  • 关键词

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