Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA.
Mass Spectrometry, Proteomics and Metabolomics Core Facility, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA.
Proteomics. 2023 Oct;23(20):e2300150. doi: 10.1002/pmic.202300150. Epub 2023 May 18.
Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.
血清无疑是用于疾病预测和诊断的最常分析的生物液体。在此，我们使用基于下拉式蛋白质组学的方法，针对五种不同的血清丰富蛋白耗竭 (SAPD) 试剂盒，评估了其在鉴定人类血清中疾病特异性生物标志物方面的性能。不出所料，SAPD 试剂盒的 IgG 去除效率差异很大，范围从 70%到 93%。数据库搜索结果的两两比较显示，试剂盒之间的蛋白鉴定存在 10%-19%的差异。针对 IgG 和白蛋白的基于免疫捕获的 SAPD 试剂盒在去除这两种丰富蛋白方面优于其他试剂盒。相反，基于非抗体的方法（即使用离子交换树脂的试剂盒）和利用多抗体方法的试剂盒在从样品中去除 IgG/白蛋白方面证明效率较低，但可鉴定的肽数量最多。值得注意的是，与未耗尽的样本相比，我们的结果表明，不同的癌症生物标志物可根据所使用的 SAPD 试剂盒的不同而富集高达 10%。此外，对下拉式蛋白质组学结果的功能分析表明，不同的 SAPD 试剂盒富集了不同的疾病和途径特异性蛋白质组。总的来说，我们的研究强调，在通过鸟枪法蛋白质组学分析血清中的疾病生物标志物时，仔细选择合适的商业 SAPD 试剂盒至关重要。
