We evaluate here the combination of two-dimensional liquid chromatography (2D-LC) in the multiple heart cutting mode and isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples. As a proof of concept, we attempt the quantification of proteotypic peptides of Apolipoprotein AIV (APOA4), Complement C3 (C3) and Vitronectin (VTN) which have been previously identified as potential candidate biomarkers of glaucoma. Using this 2D-LC strategy, analyte enrichment steps are avoided and the sample preparation involved after enzymatic digestion amounted to a simple centrifugation, evaporation of the supernatant and reconstitution in the 1D mobile phase. A mobile phase not compatible with the ESI source (10 mM KH₂PO₄ at pH 2.7) was used in the first dimension as it provided a satisfactory chromatographic resolution of the peptides and a high buffering capacity avoiding changes in retention times when analyzing complex matrices like human serum. We also demonstrate that using coeluting labelled analogues of the target peptides, protein concentrations were not affected by slight retention time shifts affecting the amount of target peptides transferred to the second dimension. Satisfactory results were obtained when analyzing fortified serum samples (recoveries from 98 to 113%). Precisions in the range of 1–9% RSD were obtained when replicating the analysis of a pooled serum sample. The comparative analysis of serum samples from n = 94 control subjects and n = 91 patients diagnosed with primary open-angle glaucoma did not show significant differences in the APOA4, VTN and C3 concentrations in contrast with previous studies using immunoassays.

我们在此评估了多中心切割模式下二维液相色谱 (2D-LC) 和同位素稀释串联质谱法的组合，用于直接分析血清样品的胰蛋白酶消化物。作为概念证明，我们尝试对载脂蛋白 AIV (APOA4)、补体 C3 (C3) 和玻连蛋白 (VTN) 的蛋白型肽进行量化，这些蛋白以前已被确定为青光眼的潜在候选生物标志物。使用这种 2D-LC 策略，避免了分析物富集步骤，酶消化后涉及的样品制备相当于简单的离心、上清液的蒸发和在一维流动相中的重组。与 ESI 源不兼容的流动相 (10 mM KH ₂ PO ₄pH 2.7) 用于第一维，因为它提供了令人满意的肽色谱分辨率和高缓冲能力，在分析复杂基质（如人血清）时避免了保留时间的变化。我们还证明，使用目标肽的共洗脱标记类似物，蛋白质浓度不受轻微保留时间偏移的影响，这些保留时间偏移会影响转移到第二维的目标肽的数量。在分析强化血清样品时获得了令人满意的结果（回收率为 98% 至 113%）。重复分析混合血清样品时，获得了 1–9% RSD 范围内的精密度。