Pharmacology & Transplantation (P&T), Université de Limoges, INSERM U1248, Limoges 87000, France.
Department of Pharmacology, Toxicology and Pharmacovigilance, CHU Limoges, Limoges 87000, France.
Anal Chem. 2024 Jan 16;96(2):746-755. doi: 10.1021/acs.analchem.3c03933. Epub 2024 Jan 2.
Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.
使用可靠的定量方法对特征明确的生物标志物对于各种病理情况的管理至关重要，例如糖尿病、肾脏和肝脏疾病。人血清白蛋白（HSA）同工型作为先进肝脏病理的生物标志物越来越受到关注。鉴于 HSA 观察到的结构改变，需要深入了解其同工型，以将其确立为可靠的生物标志物。因此，似乎需要一种稳健的绝对定量方法。在这项研究中，我们开发并验证了一种更先进的自上而下的液相色谱-质谱（LC-MS）方法，用于绝对定量 HSA 同工型，使用肌红蛋白（Mb）作为定量和质量重新校准的内标。基于解卷积光谱和提取离子色谱（XIC）的峰积分研究了两种不同的定量方法。HSA/肌红蛋白蛋白混合物以良好分离的峰形式洗脱。Mb 允许对每个样品进行系统的质量重新校准，与传统的基于解卷积的方法相比，质量偏差极低。总共定量了 8 种感兴趣的 HSA 同工型。通过使用解卷积峰获得了具有良好线性的特定同工型校准曲线。值得注意的是，HSA 的电离行为似乎依赖于同工型，这表明需要使用富含同工型的溶液作为校准标准，用于 HSA 同工型的绝对定量研究。观察到良好的重复性、重现性和准确性，与常规生化测定相比，对白蛋白浓度较低的样品具有更好的灵敏度。通过相对简单的工作流程，该方法在绝对定量中的应用具有很大的潜力，特别是在需要高通量方法和灵敏度的临床环境中进行 HSA 同工型研究时。
