Accurate quantification of low-abundance protein Cystatin-C (CysC) in serum by liquid chromatography tandem mass spectrometry (LC–MS/MS) method is very difficult. After sample processing and tryptic digestion, the matrix of CysC surrogate peptides is very complexity, and the concentrations of them are very low, so solid-phase extraction (SPE) must be used to make the surrogate peptides purification and enrichment. In this paper, we used C18 reversed-phase SPE (RP-SPE) and mixed-mode SPE as SPE cartridges. We quantitatively assessed and compared the CysC surrogate peptides recoveries and matrix effects by different SPE cartridges. The sequence of CysC surrogate peptide is ALDFAVGEYNK, and sequence-specific subions y6 (VGEYNK+, m/z 709.3) and y9 (DFAVGEYNK+, m/z 1042.4) were selected for quantification of CysC, because the two fragment ions showed the highest sensitivity. In neat solution, the highest recoveries were similarly for y9 and y6 when used RP-SPE and mixed-mode SPE. However, in serum matrix, the recoveries were significantly higher when used mixed-mode SPE than RP-SPE, which was caused by matrix effects. Results showed that both RP-SPE and mixed-mode SPE were resulted in ion enhancement for CysC surrogate peptides quantification by LC–MS/MS, but mixed-mode SPE reduced more matrix effects. So mixed-mode SPE was more suitable SPE type for purification and enrichment of CysC surrogate peptides.

中文翻译：

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反相和混合模式 SPE 对 CysC 中替代肽富集和净化的比较，通过 LC-MS/MS 定量

通过液相色谱串联质谱法 (LC–MS/MS) 方法准确定量血清中的低丰度蛋白胱抑素 C (CysC) 非常困难。经过样品处理和胰蛋白酶消化后，CysC替代肽的基质非常复杂，浓度很低，因此必须采用固相萃取（SPE）对替代肽进行纯化和富集。在本文中，我们使用 C18 反相 SPE (RP-SPE) 和混合模式 SPE 作为 SPE 小柱。我们通过不同的 SPE 小柱定量评估和比较了 CysC 替代肽的回收率和基质效应。CysC 替代肽的序列是 ALDFAVGEYNK，选择序列特异性亚离子 y6（VGEYNK+，m/z 709.3）和 y9（DFAVGEYNK+，m/z 1042.4）用于 CysC 的定量，因为两个碎片离子表现出最高的灵敏度。在纯溶液中，当使用 RP-SPE 和混合模式 SPE 时，y9 和 y6 的回收率最高。然而，在血清基质中，使用混合模式 SPE 时的回收率明显高于 RP-SPE，这是由基质效应引起的。结果表明，RP-SPE 和混合模式 SPE 均导致通过 LC-MS/MS 定量 CysC 替代肽的离子增强，但混合模式 SPE 减少了更多的基质效应。因此，混合模式 SPE 是更适合纯化和富集 CysC 替代肽的 SPE 类型。这是由矩阵效应引起的。结果表明，RP-SPE 和混合模式 SPE 均导致通过 LC-MS/MS 定量 CysC 替代肽的离子增强，但混合模式 SPE 减少了更多的基质效应。因此，混合模式 SPE 是更适合纯化和富集 CysC 替代肽的 SPE 类型。这是由矩阵效应引起的。结果表明，RP-SPE 和混合模式 SPE 均导致通过 LC-MS/MS 定量 CysC 替代肽的离子增强，但混合模式 SPE 减少了更多的基质效应。因此，混合模式 SPE 是更适合纯化和富集 CysC 替代肽的 SPE 类型。