W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
Anal Chem. 2010 Dec 15;82(24):10179-85. doi: 10.1021/ac102248d. Epub 2010 Nov 19.
Biomarker discovery efforts in serum and plasma are greatly hindered by the presence of high abundance proteins that prevent the detection and quantification of less abundant, yet biologically significant, proteins. The most common method for addressing this problem is to specifically remove the few abundant proteins through immunoaffinity depletion/subtraction. Herein, we improved upon this method by utilizing multiple depletion columns in series, so as to increase the efficiency of the abundant protein removal and augment the detection/identification of less abundant plasma proteins. Spectral counting was utilized to make quantitative comparisons between undepleted plasma, plasma depleted with a single depletion column, and plasma depleted using two or three depletion columns in tandem. In the undepleted plasma only 29 lower abundance protein groups were identified with the top-scoring protein from each group having a median spectral count of 3, while in the plasma processed using a single HSA depletion column 61 such protein groups were identified with a median spectral count of 8. In comparison, 76 lesser abundant protein groups were identified with a median spectral count of 11.5 in the two column setup (i.e., HSA followed by MARS Hu14). However, in the ultimate depleted plasma sample, which was created using three depletion columns in tandem, the number of less abundant protein groups identified increase to 81 and the median spectral count for the top-scoring proteins from each group increased to 15 counts per protein. Moreover, exogenous B-type natriuretic peptide-32, which was added to the plasma as a detection benchmark at 12 μg/mL, was only detected in the plasma sample depleted using three depletion columns in tandem. Collectively, these data demonstrate that this method, tandem removal of abundant proteins or TRAP, provides superior removal efficiency compared to traditional applications and improves the depth of proteome coverage in plasma.
在血清和血浆中进行生物标志物发现的工作受到高丰度蛋白质的存在极大阻碍，这些蛋白质会阻止检测和定量分析较少丰度但具有重要生物学意义的蛋白质。解决这个问题最常用的方法是通过免疫亲和去除/减去特异性去除少数丰富的蛋白质。在此，我们通过串联使用多个去除柱来改进该方法，以提高丰富蛋白质去除的效率并增加较少丰度血浆蛋白质的检测/鉴定。谱计数用于对未处理的血浆、用单个去除柱处理的血浆和串联使用两个或三个去除柱处理的血浆进行定量比较。在未处理的血浆中，仅鉴定到 29 个低丰度蛋白质组，每个组的最高得分蛋白的中位数谱计数为 3，而在使用单个 HSA 去除柱处理的血浆中，鉴定到 61 个这样的蛋白质组，中位数谱计数为 8。相比之下，在两柱设置（即 HSA 后接 MARS Hu14）中，鉴定到 76 个低丰度蛋白质组，中位数谱计数为 11.5。然而，在使用三个串联去除柱创建的最终耗尽的血浆样本中，鉴定到的低丰度蛋白质组数量增加到 81，每个组的最高得分蛋白的中位数谱计数增加到每个蛋白 15 个计数。此外，作为检测基准添加到血浆中的外源性 B 型利钠肽-32（12μg/mL）仅在使用三个串联去除柱的血浆样本中被检测到。总之，这些数据表明，与传统应用相比，这种方法（即丰度蛋白的串联去除或 TRAP）提供了更高的去除效率，并提高了血浆中蛋白质组覆盖的深度。
