Background B-cell maturation antigen is a pivotal therapeutic target for multiple myeloma (MM). Membrane-bound BCMA can be cleaved by γ-secretase and shed as soluble BCMA (sBCMA). sBCMA can act as a neutralizing sink to compete with drug, as well as serve as a diagnostic/prognostic biomarker for MM. Antibody-capture based methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoaffinity-liquid chromatography-multiple reaction monitoring (IA-LC-MRM), have been reported and well adopted to measure sBCMA in clinical samples. However, both methods are biased by capturing antibodies. Methods We have used various LC-MS workflows to characterize and quantify endogenous sBCMA in MM patient samples, including bottom-up peptide mapping, intact analysis, IA-based, and reagent-free (RF)-LC-MRM quantitation. Results We have confirmed that sBCMA contains a variable N-terminus and a C-terminus that extends to the transmembrane domain, ending at amino acid 61. Leveraging an in-house synthesized G-1-61 sBCMA recombinant standard, we developed a RF-LC-MRM method for unbiased sBCMA quantitation in MM patient samples. By comparing the results from RF-LC-MRM with ELISA and IA-LC-MRM, we demonstrated that RF-LC-MRM measures a more complete pool of endogenous sBCMA compared to the antibody-based methods. Conclusions This work fills the knowledge gap of the exact sequence of endogenous sBCMA for the first time, which differs from the current commercially available standard. Additionally, this work highlights the necessity of identifying the actual sequence of an endogenous soluble target such as sBCMA, both for bioanalytical purposes and to underpin pharmacodynamic measurements.

中文翻译：

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通过 LC-MS 破译多发性骨髓瘤患者样本中内源性可溶性 B 细胞成熟抗原的精确序列和无偏定量

背景 B 细胞成熟抗原是多发性骨髓瘤 (MM) 的关键治疗靶点。膜结合的 BCMA 可以被 γ-分泌酶裂解并以可溶性 BCMA (sBCMA) 的形式脱落。sBCMA 可以作为中和池与药物竞争，也可以作为 MM 的诊断/预后生物标志物。基于抗体捕获的方法，例如酶联免疫吸附测定 (ELISA) 和免疫亲和-液相色谱-多反应监测 (IA-LC-MRM)，已被报道并广泛用于测量临床样本中的 sBCMA。然而，这两种方法都因捕获抗体而存在偏差。方法 我们使用各种 LC-MS 工作流程来表征和量化 MM 患者样本中的内源 sBCMA，包括自下而上的肽图谱、完整分析、基于 IA 和无试剂 (RF)-LC-MRM 定量。结果 我们已经证实 sBCMA 包含一个可变的 N 端和一个延伸至跨膜结构域的 C 端，终止于氨基酸 61。利用内部合成的 G-1-61 sBCMA 重组标准品，我们开发了 RF-用于 MM 患者样本中无偏 sBCMA 定量的 LC-MRM 方法。通过将 RF-LC-MRM 与 ELISA 和 IA-LC-MRM 的结果进行比较，我们证明，与基于抗体的方法相比，RF-LC-MRM 可以测量更完整的内源 sBCMA 池。结论本工作首次填补了内源性sBCMA精确序列的知识空白，与目前的市售标准不同。此外，这项工作强调了识别内源性可溶性靶标（例如 sBCMA）的实际序列的必要性，既用于生物分析目的，又支持药效学测量。