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Utilizing Spectral Counting To Quantitatively Characterize Tandem Removal of Abundant Proteins (TRAP) in Human Plasma

机译:利用光谱计数来定量表征串联分离血浆中大量蛋白质(TRAP)

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摘要

Biomarker discovery efforts in serum and plasma arengreatly hindered by the presence of high abundancenproteins that prevent the detection and quantification ofnless abundant, yet biologically significant, proteins. Thenmost common method for addressing this problem is tonspecifically remove the few abundant proteins throughnimmunoaffinity depletion/subtraction. Herein, we improvednupon this method by utilizing multiple depletionncolumns in series, so as to increase the efficiency of thenabundant protein removal and augment the detectionidentification of less abundant plasma proteins. Spectralncounting was utilized to make quantitative comparisonsnbetween undepleted plasma, plasma depleted with ansingle depletion column, and plasma depleted using twonor three depletion columns in tandem. In the undepletednplasma only 29 lower abundance protein groups werenidentified with the top-scoring protein from each groupnhaving a median spectral count of 3, while in the plasmanprocessed using a single HSA depletion column 61 suchnprotein groups were identified with a median spectralncount of 8. In comparison, 76 lesser abundant proteinngroups were identified with a median spectral count ofn11.5 in the two column setup (i.e., HSA followed by MARSnHu14). However, in the ultimate depleted plasma sample,nwhich was created using three depletion columns inntandem, the number of less abundant protein groupsnidentified increase to 81 and the median spectral countnfor the top-scoring proteins from each group increasednto 15 counts per protein. Moreover, exogenous B-typennatriuretic peptide-32, which was added to the plasmanas a detection benchmark at 12 μg/mL, was only detectednin the plasma sample depleted using three depletionncolumns in tandem. Collectively, these data demonstratenthat this method, tandem removal of abundant proteinsnor TRAP, provides superior removal efficiency comparednto traditional applications and improves the depth ofnproteome coverage in plasma.
机译:高丰度蛋白的存在阻碍了血清和血浆中生物标志物发现的努力,高丰度蛋白阻止了对丰富而生物学上重要的蛋白质的检测和定量。解决此问题的最常用方法是通过免疫亲和力耗竭/扣除来特异性去除少数丰富的蛋白质。在本文中,我们通过串联使用多个耗尽柱改进了这种方法,从而提高了去除大量蛋白质的效率,并增强了对血浆蛋白含量较低的检测/鉴定。光谱计数用于在未耗尽血浆,使用单耗尽柱消耗的血浆和使用两个或三个耗尽柱串联消耗的血浆之间进行定量比较。在未耗尽血浆中,只有29个较低丰度的蛋白质组被鉴定为每个组中得分最高的蛋白质,其光谱中位数为3,而在使用单个HSA耗尽柱处理的血浆中,鉴定出61个此类蛋白质组,其光谱中位数为8。在两个色谱柱设置中(即HSA,然后是MARSnHu14),鉴定出76个较少的丰富蛋白质组,其平均光谱计数为n11.5。但是,在最终消耗的血浆样品中(使用三个耗尽柱同时产生),识别出的较不丰富的蛋白质组数量增加到81个,每组中得分最高的蛋白质的中值光谱计数n增加到每个蛋白质15个计数。此外,仅在使用三个串联消耗柱的消耗的血浆样品中检测到外源性B型钠尿尿素肽32(其添加到血浆中的检测基准为12μg/ mL)。总的来说,这些数据表明,与传统应用相比,这种既能富集蛋白质又不会TRAP的串联去除方法提供了优越的去除效率,并提高了蛋白质组在血浆中的覆盖深度。

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  • 来源
    《Analytical Chemistry》 |2010年第24期|p.10179-10185|共7页
  • 作者单位

    W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University,Raleigh, North Carolina 27695, United States, and Division for Cardiovascular Disease, Mayo Clinic College ofMedicine, Rochester, Minnesota 55905, United States;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 入库时间 2022-08-17 13:36:53

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