Benchmarking Cleavable Biotin Tags for Peptide-Centric Chemoproteomics

Click chemistryspecifically
the copper-catalyzed azide-alkyne
cycloadditionhas enabled the development of a wide range of
chemical probes to analyze subsets of the functional proteome. The
“clickable” proteome can be selectively enriched by
using diverse cleavable biotin tags, but the direct identification
of probe/tag-modified peptides (or peptide-centric analysis) remains
challenging. Here, we evaluated the performance of five commercially
available cleavable biotin tags in three most common chemoproteomic
workflows, resulting in a comparative analysis of 15 methods. An acid-cleavable
biotin tag with a dialkoxydiphenylsilane moiety, in combination with
the protein “click”, peptide “capture”
workflow, outperforms all other methods in terms of enrichment efficiency,
identification yield, and reproducibility, although its performance
may be slightly compromised by the formation of an unwanted formate
product revealed by blind search. Despite being inferior, photocleavable,
and reduction-cleavable, biotin tags can also find their applicable
sceneries, especially when dealing with acid-sensitive probes or probe-derived
modifications. Furthermore, the systematic comparison of LC–MS/MS
characteristics of tag-modified peptides provides valuable information
for the future development of cleavable biotin reagents. Taken together,
our data provides a much-needed practical guidance for researchers
interested in developing and/or applying an ideal cleavable biotin
tag to peptide-centric chemoproteomics.

点击化学（Click chemistry）——具体为铜催化叠氮-炔环加成反应（copper-catalyzed azide-alkyne cycloaddition）——已推动开发出一系列化学探针，用于解析功能蛋白质组的特定亚群。借助多样化的可裂解生物素标签（cleavable biotin tags），可对“可点击”蛋白质组实现选择性富集，但直接鉴定探针/标签修饰肽段（以肽段为中心的分析，peptide-centric analysis）仍存在较大挑战。本研究针对五种商用可裂解生物素标签，在三种最常用的化学蛋白质组学工作流程中的表现进行了评估，最终完成对15种方法的对比分析。带有二烷氧基二苯硅基结构域（dialkoxydiphenylsilane moiety）的酸裂解型生物素标签（acid-cleavable biotin tag），结合蛋白质“点击”、肽段“捕获”的工作流程，在富集效率、鉴定产率与重现性方面均优于其余所有方法；不过盲搜分析显示，该标签会生成非目标甲酸副产物，可能会小幅削弱其性能表现。尽管性能稍逊，光裂解型（photocleavable）与还原裂解型（reduction-cleavable）生物素标签仍有适用场景，尤其在处理酸敏感探针或探针衍生修饰时优势明显。此外，本研究对标签修饰肽段的液相色谱-串联质谱（LC–MS/MS）特征开展系统性对比，可为未来可裂解生物素试剂的开发提供宝贵参考信息。综上，本研究数据可为有意开发或应用理想可裂解生物素标签以开展以肽段为中心的化学蛋白质组学（chemoproteomics）研究的科研人员提供亟需的实用指导。