Asicioglu Meltem, Swart Claudia, Saban Evren, Yurek Emrah, Karaguler Nevin Gul, Oztug Merve
70777 TUBITAK National Metrology Institute (TUBITAK UME) , Kocaeli, Türkiye.
Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Türkiye.
Clin Chem Lab Med. 2025 Jan 3;63(5):1016-1030. doi: 10.1515/cclm-2024-0999. Print 2025 Apr 28.
An analytical protocol based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), which includes a peptide-based calibration strategy, was developed and validated for the determination of cardiac troponin I (cTnI) levels in clinical samples. Additionally, the developed method was compared with a protein-based calibration strategy, using cTnI serving as a model for low-abundant proteins. The aim is to evaluate new approaches for protein quantification in complex matrices, supporting the metrology community in implementing new methods and developing fit-for-purpose SI- traceable peptide or protein primary calibrators.
To establish traceability to SI units, peptide impurity correction amino acid analysis (PICAA) was conducted to determine the absolute content of signature peptides in the primary standards. Immunoaffinity enrichment was used to capture cTnI from human serum, with a comparison between microbeads and nanobeads to improve enrichment efficiency. Parallel reaction monitoring was used to monitor two signature peptides specific to cTnI. Various digestion parameters were optimized to achieve complete digestion.
The analytical method demonstrated selectivity and specificity, allowing the quantification of cTnI within 0.9-22.0 μg/L. The intermediate precision RSD was below 28.9 %, and the repeatability RSD was below 5.8 % at all concentration levels, with recovery rates ranging from 87 % to 121 %. The comparison of calibration strategies showed similar LOQ values, but the peptide-based calibration exhibited significant quantitative bias in recovery rates. The data are available via ProteomeXchange (PXD055104).
This isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method, based on peptide calibration, successfully quantified cTnI in human serum. Comparing this with protein-based calibration highlighted both the strengths and potential limitations of peptide-based strategies.
开发并验证了一种基于同位素稀释液相色谱 - 串联质谱法（ID-LC-MS/MS）的分析方案，该方案包括基于肽的校准策略，用于测定临床样本中心肌肌钙蛋白I（cTnI）的水平。此外，以cTnI作为低丰度蛋白的模型，将所开发的方法与基于蛋白的校准策略进行了比较。目的是评估复杂基质中蛋白质定量的新方法，支持计量学界实施新方法并开发适用于目的的国际单位制（SI）可溯源肽或蛋白质一级校准品。
为建立与SI单位的溯源性，进行了肽杂质校正氨基酸分析（PICAA）以确定一级标准品中特征肽的绝对含量。采用免疫亲和富集从人血清中捕获cTnI，比较了微珠和纳米珠以提高富集效率。采用平行反应监测来监测cTnI特有的两种特征肽。优化了各种消化参数以实现完全消化。
该分析方法具有选择性和特异性，能够在0.9 - 22.0μg/L范围内对cTnI进行定量。在所有浓度水平下，中间精密度相对标准偏差（RSD）低于28.9%，重复性RSD低于5.8%，回收率在87%至121%之间。校准策略的比较显示出类似的定量限（LOQ）值，但基于肽的校准在回收率方面表现出显著的定量偏差。数据可通过蛋白质组交换库（PXD055104）获取。
这种基于肽校准的同位素稀释液相色谱 - 串联质谱法（ID-LC-MS/MS）成功地对人血清中的cTnI进行了定量。将其与基于蛋白的校准进行比较，突出了基于肽的策略的优势和潜在局限性。
