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<p>Isotope dilution mass spectrometry (IDMS) is a critical technique for the quantification of protein biomarkers in complex biological samples, enabling sensitive measurement through integration with sample preparation to eliminate matrix interference. Nevertheless, the interchangeability between distinct pretreatment strategies is poorly defined. Herein, two traceable IDMS methods, immune mass spectrometry (IMS) and direct mass spectrometry (DMS), that combine immunoaffinity or antibody-free pretreatment were established for the quantification of plasma ProGRP, which is a recognized biomarker for small cell lung cancer. The internal standard curve was established using NLLGLIEAK, an enzymatic hydrolysis peptide of ProGRP, and its isotope-labeled molecule to ensure quantitative traceability. A microliter high-performance liquid chromatography tandem mass spectrometry platform was established for sensitive quantification of ProGRP in the range of 0.398–13.8&#xa0;ng/mL. Correction factors were introduced to eliminate the effects of protein loss and enzyme digestion efficiency on the quantitative results, which were 86.84% for IMS and 85.48% for DMS. The RSDs of IMS and DMS were 5.5% and 4.3%, respectively. The comparability among the established MS methods and two immune methods was studied using plasma from patients with abnormally high expression of ProGRP. The IMS and DMS results showed good consistency and good correlation with the results of clinical chemiluminescent immunoassay (CLIA), while the correlation between CLIA and enzyme-linked immunosorbent assay (ELISA) results was poor. The factors affecting quantification accuracy by MS analysis and immunoassay were discussed, revealing the potential of IMS and DMS as reference measurement procedures for protein biomarkers in complex matrices.</p> Graphical Abstract <p></p>

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Comparative evaluation of immunoaffinity and antibody-free isotope dilution mass spectrometry approaches for plasma ProGRP quantification

  • 期刊论文

  • 发表日期：

    2025年12月20日

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  • Jiahui Li,
  • Huan Yao,
  • Qiaoxuan Zhang,
  • Yuqi Yan,
  • Xinling Cui,
  • Xianzhang Huang,
  • Wenjing Xing,
  • Huanyu Ju,
  • Dewei Song,
  • Peng Xiao,
  • Jian Ma

摘要

Isotope dilution mass spectrometry (IDMS) is a critical technique for the quantification of protein biomarkers in complex biological samples, enabling sensitive measurement through integration with sample preparation to eliminate matrix interference. Nevertheless, the interchangeability between distinct pretreatment strategies is poorly defined. Herein, two traceable IDMS methods, immune mass spectrometry (IMS) and direct mass spectrometry (DMS), that combine immunoaffinity or antibody-free pretreatment were established for the quantification of plasma ProGRP, which is a recognized biomarker for small cell lung cancer. The internal standard curve was established using NLLGLIEAK, an enzymatic hydrolysis peptide of ProGRP, and its isotope-labeled molecule to ensure quantitative traceability. A microliter high-performance liquid chromatography tandem mass spectrometry platform was established for sensitive quantification of ProGRP in the range of 0.398–13.8 ng/mL. Correction factors were introduced to eliminate the effects of protein loss and enzyme digestion efficiency on the quantitative results, which were 86.84% for IMS and 85.48% for DMS. The RSDs of IMS and DMS were 5.5% and 4.3%, respectively. The comparability among the established MS methods and two immune methods was studied using plasma from patients with abnormally high expression of ProGRP. The IMS and DMS results showed good consistency and good correlation with the results of clinical chemiluminescent immunoassay (CLIA), while the correlation between CLIA and enzyme-linked immunosorbent assay (ELISA) results was poor. The factors affecting quantification accuracy by MS analysis and immunoassay were discussed, revealing the potential of IMS and DMS as reference measurement procedures for protein biomarkers in complex matrices.

Graphical Abstract