Krombholz Sophia, Thomas Andreas, Delahaut Philippe, Bidlingmaier Martin, Schilbach Katharina, Miller Geoffrey, Thevis Mario
Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Germany.
CER Groupe - Département Santé, Marloie, Belgium.
Growth Horm IGF Res. 2023 Oct-Dec;72-73:101560. doi: 10.1016/j.ghir.2023.101560. Epub 2023 Nov 21.
The precise and accurate quantification of human growth hormone (GH) in plasma/ serum is crucial for the diagnosis and treatment of diseases like GH deficiency or acromegaly. However, the ligand-binding assays (LBAs) currently used for routine testing show considerable methodological variability. Here, we present a complementary, combined top-down and bottom-up LC-MS-based method to quantify (intact) GH in plasma and serum, which concurrently provides a basis for a MS-based analysis of GH in doping controls.
Extraction of GH from plasma/ serum was accomplished by protein precipitation, followed by an immunocapture step using protein A-coupled magnetic beads and a polyclonal anti-GH antibody. The intact protein was subsequently analyzed top-down on a 2D-LC-HRMS/MS system. In addition, sample extracts were digested with trypsin and analyzed for signal peptides corresponding to 'total', 22 kDa and 20 kDa GH (bottom-up). Both assays were validated according to current guidelines and compared to the GH isoform differential immunoassay used in routine doping control analysis. GH concentrations in serum samples of healthy adults, patients with acromegaly, and in samples obtained after administration of recombinant GH were analyzed as proof-of-principle.
The intact monomeric 22 kDa isoform of GH was selectively quantified in a representative working range of 0.5 to 10 ng/ml by top-down LC-HRMS/MS. Subsequent bottom-up analysis provided additional data on 'total' and 20 kDa GH. Top-down and bottom-up assay results for the 22 kDa isoform correlated well with the corresponding immunoassay results (R > 0.95). For a possible application of the method in an anti-doping context, the ratio between 22 kDa and 'total' GH was evaluated, indicating differences between the various donor groups, but only with limited significance.
The top-down and bottom-up LC-HRMS/MS method developed here presents a valuable tool for the quantification of GH in plasma/ serum complementary to established LBAs used at present in clinical measurements. Albeit the examination of the GH isoform proportions by the LC-MS method does not yet allow for the assessment of GH abuse, the obtained findings provide an important basis to enable LC-MS-based GH analysis of doping control samples in the future.
准确、精确地定量血浆/血清中的人生长激素（GH）对于诊断和治疗诸如生长激素缺乏症或肢端肥大症等疾病至关重要。然而，目前用于常规检测的配体结合分析（LBA）显示出相当大的方法学变异性。在此，我们提出一种基于液相色谱-质谱联用的自上而下和自下而上相结合的补充方法，用于定量血浆和血清中的（完整）GH，同时为兴奋剂检测中基于质谱的GH分析提供基础。
通过蛋白质沉淀从血浆/血清中提取GH，随后使用蛋白A偶联磁珠和多克隆抗GH抗体进行免疫捕获步骤。随后在二维液相色谱-高分辨质谱/质谱系统上对完整蛋白质进行自上而下分析。此外,用胰蛋白酶消化样品提取物，并分析对应于“总”、22 kDa和20 kDa GH的信号肽（自下而上）。两种分析方法均根据现行指南进行验证，并与常规兴奋剂检测分析中使用的GH同工型差异免疫分析进行比较。作为原理验证，分析了健康成年人、肢端肥大症患者血清样本以及注射重组GH后获得的样本中的GH浓度。
通过自上而下的液相色谱-高分辨质谱/质谱在0.5至10 ng/ml的代表性工作范围内选择性地定量了完整的22 kDa单体同工型GH。随后的自下而上分析提供了关于“总”GH和20 kDa GH的额外数据。22 kDa同工型的自上而下和自下而上分析结果与相应的免疫分析结果相关性良好（R>0.95）。对于该方法在反兴奋剂背景下的可能应用，评估了22 kDa与“总”GH之间的比率，表明不同供体组之间存在差异，但意义有限。
这里开发的自上而下和自下而上的液相色谱-高分辨质谱/质谱方法是一种有价值的工具，可用于定量血浆/血清中的GH，作为目前临床测量中使用的既定LBA的补充。尽管通过液相色谱-质谱法检查GH同工型比例尚不能评估GH滥用情况，但所获得的结果为未来基于液相色谱-质谱的兴奋剂检测样本GH分析提供了重要基础。
