Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC-MS/MS analysis. LC-MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the "waste" fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded "waste" fractions contain proteins of potential interest and should be included in CSF biomarker studies.

中文翻译：

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亲和力耗竭与相对蛋白质富集：两种增加人脑脊髓液蛋白质组覆盖率的主要策略的并排比较。

脑脊液（CSF）与中枢神经系统直接接触。这使得人脑脊液成为神经系统疾病潜在生物标志物的诱人来源。与血浆相似，CSF的蛋白质组学分析由于单个蛋白质浓度的高动态范围以及几种高度丰富的蛋白质的存在而变得复杂。为了处理丰富的人类CSF蛋白，通常使用针对血浆/血清开发的方法。使用组合肽配体库的多重亲和力去除系统和蛋白质含量较低的蛋白质富集是最常见的方法。但是，它们对CSF蛋白质组覆盖率的相对影响从未在单个研究中同时进行评估。因此，我们探索了使用MARS 14柱和ProteoMiner配体库对CSF耗竭对随后LC-MS / MS分析中鉴定的CSF蛋白数量的影响。粗制（未经处理）CSF的LC-MS / MS分析提供了大约500种鉴定出的蛋白质。MARS 14滤芯消耗的CSF将鉴定的数量增加到将近800，而使用ProteoMiner处理CSF则可以鉴定600的蛋白质。为了探索在耗竭过程中CSF蛋白的潜在损失，我们还分析了两种方法产生的“废物”级分，即MARS 14弹药筒保留的蛋白，以及ProteoMiner的流通级分中存在的分子。超过250种蛋白质与MARS 14弹药筒结合，其中100种蛋白质未在相应的耗尽CSF中鉴定。同样，在ProteoMiner工作流程中对废物级分进行的分析提供了在配体库耗尽的CSF中找不到的近70种独特蛋白质。与粗略的CSF相比，两种耗竭策略均显着增加了已鉴定CSF蛋白的数量。但是，与ProteoMiner（611）相比，MARS 14耗竭提供了明显更多的鉴定蛋白质（773）。此外，我们显示，在耗损过程中，由于共耗损（MARS 14）或排斥（ProteoMiner），导致CSF蛋白丢失。这表明常规丢弃的“废物”级分包含潜在感兴趣的蛋白质，应包括在CSF生物标志物研究中。与粗略的CSF相比，两种耗竭策略均显着增加了已鉴定CSF蛋白的数量。但是，与ProteoMiner（611）相比，MARS 14耗竭提供了明显更多的鉴定蛋白质（773）。此外，我们显示，在耗损过程中，由于共耗损（MARS 14）或排斥（ProteoMiner），导致CSF蛋白丢失。这表明常规丢弃的“废物”级分包含潜在感兴趣的蛋白质，应包括在CSF生物标志物研究中。与粗略的CSF相比，两种清除策略均显着增加了已鉴定的CSF蛋白的数量。但是，与ProteoMiner（611）相比，MARS 14耗竭提供了明显更多的鉴定蛋白质（773）。此外，我们显示，在耗损过程中，由于共耗损（MARS 14）或排斥（ProteoMiner），导致CSF蛋白丢失。这表明常规丢弃的“废物”级分包含潜在感兴趣的蛋白质，应包括在CSF生物标志物研究中。