Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma
Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma
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DOI：
10.1002/pmic.200401228
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发表时间：
2005-08-01
期刊：
影响因子：
3.4
通讯作者：
Speicher, DW
中科院分区：
文献类型：
--
作者：
Echan, LA;Tang, HY;Speicher, DW
系统检测人类血液中可能是推定疾病生物标志物的低丰度蛋白质，由于蛋白质丰度范围极广而变得复杂。因此，主要蛋白质的消耗是用于增强血清或血浆中的检测灵敏度的一种潜在策略。本研究比较了最近商业化的HPLC柱，该柱含有针对六种最丰富的血液蛋白质的抗体（“Top-6耗尽”），使用较旧的Cibacron蓝/蛋白A或G耗尽方法或不耗尽。此外，还评价了HPLC柱的原型旋转柱版本和替代原型双抗体旋转柱。HPLC多克隆抗体柱及其旋转柱版本是非常有前途的方法，用于大幅简化人血清或血浆样品。这些微柱显示了检测的消除方法的最低非特异性结合。相比之下，其他亲和方法，特别是基于染料的树脂，产生了许多蛋白质的结合级分，除了目标蛋白质。六种丰富蛋白质的消耗从人血清或血浆中去除了约85%的总蛋白质，这使得10- 20倍更高量的消耗的血清或血浆样品被应用于2-D凝胶或替代蛋白质分析方法，如蛋白质阵列像素化。然而，在2-D凝胶上检测到的新斑点的数量是适度的，并且大多数新观察到的斑点是相对丰富的蛋白质的次要形式。无法检测到接近预期2-D染色极限的低丰度蛋白质可能是由于大多数血浆或血清蛋白质的高度异质性以及下一系列最丰富蛋白质对许多低丰度蛋白质的掩蔽。因此，非2-D的方法，如蛋白质阵列像素化是更有前途的策略，用于检测低丰度的蛋白质后，耗尽的六个丰富的蛋白质。
Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.