Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins
Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins
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DOI：
10.3390/ijms21165903
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发表时间：
2020-08-01
影响因子：
5.6
通讯作者：
Beck, Hans Christian
中科院分区：
文献类型：
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作者：
Palstrom, Nicolai Bjodstrup;Rasmussen, Lars Melholt;Beck, Hans Christian
在本研究中，我们评估了基于琼脂糖固定的苯甲脒（阿坝）、O-磷酸-L-酪氨酸（pTYR）、8-氨基-己基-cAMP（cAMP）或8-氨基-己基-ATP（ATP）的四种基于小分子亲和性的探针从血浆样品中去除高丰度蛋白质（如血清白蛋白）的能力，从而使得能够通过基于质谱的蛋白质组学检测血浆样品中的中低丰度蛋白质。我们将其性能与最常用的免疫耗竭方法进行了比较，即针对前14种最丰富的血浆蛋白的多亲和力去除系统Human 14（MARS 14）以及通过无标记定量液相色谱串联质谱（LC-MSMS）分析的ProteoMiner蛋白均衡方法。与多亲和力去除系统（MARS）14和Proteominer方法相比，基于亲和力的探针对低丰度血浆蛋白表现出高重现性，低至皮摩尔/mL水平，并且还表现出对大多数高丰度血浆蛋白的上级去除。就分析的蛋白质数量而言，基于ABA的亲和探针和Proteominer蛋白质均衡方法的表现优于所有其他方法。通过对6个重复实验进行相关性分析，所有检测方法对高丰度血浆蛋白和低丰度蛋白的重现性均较高。总之，我们的结果表明，基于小分子的亲和力探针是血浆蛋白质组生物标志物发现研究中常用免疫耗竭方法的绝佳替代方案。数据可通过ProteomeXchange获得，标识符为PXD 020727。
In the present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the most commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MSMS) analysis. The affinity-based probes demonstrated a high reproducibility for low-abundant plasma proteins, down to picomol per mL levels, compared to the Multi Affinity Removal System (MARS) 14 and the Proteominer methods, and also demonstrated superior removal of the majority of the high-abundant plasma proteins. The ABA-based affinity probe and the Proteominer protein equalization method performed better compared to all other methods in terms of the number of analyzed proteins. All the tested methods were highly reproducible for both high-abundant plasma proteins and low-abundant proteins as measured by correlation analyses of six replicate experiments. In conclusion, our results demonstrated that small-molecule based affinity-based probes are excellent alternatives to the commonly used immune-depletion methods for proteomic biomarker discovery studies in plasma. Data are available via ProteomeXchange with identifier PXD020727.