Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.
J Proteome Res. 2013 Jan 4;12(1):412-20. doi: 10.1021/pr300751j. Epub 2012 Dec 12.
In this paper, we have used a newly developed immunocapture and LC-MS method to demonstrate for the first time the presence of protein isoforms 1 and 3 of the small cell lung cancer (SCLC) marker progastrin-releasing peptide (ProGRP) in sera. In addition, the method allows for indirect determination of the relative presence of the other known isoform of ProGRP, also known as ProGRP isoform 2. This new method is able to determine total ProGRP as a marker in sera at clinically relevant levels and to differentiate between isoforms at the low-pM level through combining selective sample preparation by immunoextraction, tryptic digestion, and separation followed by detection with LC-SRM-MS of the signature peptides, NLLGLIEAK (total ProGRP), LSAPGSQR (ProGRP isoform 1), and DLVDSLLQVLNVK (ProGRP isoform 3), with accuracies ≤ 25% for lower limit of quantification (LLOQ) and precisions ≤ 33%. By analyzing serum samples from four patients diagnosed with SCLC using the here described new and fully validated method, the ability is shown to both determine total ProGRP concentration and to differentiate between ProGRP isoforms 1 and 3 in one single run. Quantification of various ProGRP isoforms in one single run may be helpful for further understanding of the underlying biochemical processes in SCLC and differentiation of small cell lung cancer.
在本文中，我们使用新开发的免疫捕获和 LC-MS 方法，首次证明小细胞肺癌（SCLC）标志物胃泌素释放肽前体（ProGRP）的蛋白同工型 1 和 3 存在于血清中。此外，该方法允许间接确定其他已知同工型 ProGRP（也称为 ProGRP 同工型 2）的相对存在。这种新方法能够在临床上相关的水平上确定血清中的总 ProGRP 作为标志物，并通过组合选择性免疫提取、胰蛋白酶消化和分离，然后通过 LC-SRM-MS 检测特征肽 NLLGLIEAK（总 ProGRP）、LSAPGSQR（ProGRP 同工型 1）和 DLVDSLLQVLNVK（ProGRP 同工型 3），在低 pM 水平上区分同工型，其准确度 ≤ 25%的定量下限（LLOQ）和 ≤ 33%的精密度。通过使用这里描述的新的和完全验证的方法分析四个诊断为 SCLC 的患者的血清样本，展示了在单次运行中既能确定总 ProGRP 浓度，又能区分 ProGRP 同工型 1 和 3 的能力。在单次运行中对各种 ProGRP 同工型进行定量可能有助于进一步了解 SCLC 的潜在生化过程并区分小细胞肺癌。
