Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany.
Proteomics Clin Appl. 2013 Dec;7(11-12):794-801. doi: 10.1002/prca.201300034. Epub 2013 Oct 31.
We investigated different sample pretreatment strategies and developed a standardized sample pretreatment protocol for absolute quantification of seven apolipoproteins (Apos) in human serum by LC-MS/MS using proteotypic peptides and corresponding stable isotope-labeled peptides as internal standards.
Micro-LC was coupled with quadrupole-linear ion trap MS for quantification and peptide confirmation. Denaturation, reduction, alkylation, and tryptic digestion including ultrasound and microwave assistance were investigated. Method comparison of 50 plasma samples with an immunoassay was performed for Apo A-I and Apo B.
Tryptic digestion times ranged between 5 min (Apo A-I, Apo E, Apo A-IV) and 16 h (Apo A-II). Ultrasound and microwave assistance did not improve the digestion yield. Linearity was found between 0.1 nmol/L and 100 mmol/L. The lower limits of quantification were ≤ 0.4 μmol/L for Apo A-I, Apo A-IV, Apo B-100, Apo C-I, Apo C-III, Apo E, and <1.4 μmol/L for Apo A-II. CV <13% were determined. Comparison with immunoassays showed a good agreement for Apo A-I and Apo B.
The validated preanalytical protocol enables a reliable simultaneous analysis of seven Apos in human serum without depletion. The method can now be applied in clinical studies to investigate the Apo distributions in cardiovascular diseases.
我们研究了不同的样品预处理策略，并开发了一种标准化的样品预处理方案，用于通过 LC-MS/MS 使用蛋白特征肽和相应的稳定同位素标记肽作为内标对人血清中的七种载脂蛋白（Apos）进行绝对定量。
微 LC 与四极杆线性离子阱 MS 耦合进行定量和肽确认。研究了变性、还原、烷基化和胰蛋白酶消化，包括超声和微波辅助。对 50 个血浆样本进行了免疫测定法的方法比较，用于 Apo A-I 和 Apo B。
胰蛋白酶消化时间在 5 分钟（Apo A-I、Apo E、Apo A-IV）和 16 小时（Apo A-II）之间。超声和微波辅助并没有提高消化效率。在线性范围内为 0.1 nmol/L 至 100 mmol/L。Apo A-I、Apo A-IV、Apo B-100、Apo C-I、Apo C-III、Apo E 的定量下限为 ≤0.4 μmol/L，Apo A-II 的定量下限为 <1.4 μmol/L。CV <13%。与免疫测定法的比较表明，Apo A-I 和 Apo B 具有良好的一致性。
验证的预分析方案能够可靠地同时分析人血清中的七种 Apos，而无需消耗。该方法现在可以应用于临床研究，以研究心血管疾病中 Apo 的分布。
