Isobaric Tagging-Based Selection and Quantitation of Cerebrospinal Fluid Tryptic Peptides with Reporter Calibration Curves

In the past few years, mass spectrometry (MS) has emerged as an efficient tool for the multiplexed peptide and protein concentration determination by isotope dilution. Despite the growing use of isobaric tagging to perform relative quantitation for the discovery of potential biomarkers in biological fluids, no real application has so far been presented for their absolute quantitation. Isobaric tandem mass tags (TMTs) were used herein for the selection and quantitation of tryptic peptides derived from brain damage related proteins in cerebrospinal fluid (CSF). Proteotypic tryptic peptide analogues were synthesized, prepared in four reference amounts, differentially labeled with four isobaric TMTs with reporter-ions at m/z = 128.1, 129.1, 130.1, and 131.1, and mixed with CSF sample previously labeled with TMT 126.1. Off-gel electrophoresis (OGE) was used as first-dimension separation of the pooled sample. The resulting fractions were analyzed with reversed-phase liquid chromatography (RP-LC) tandem mass spectrometry (MS/MS), using tandem time-of-flight (TOF/TOF) and hybrid linear ion trap−orbitrap (LTQ−OT) instruments. Under collision-induced dissociation (CID) or higher-energy C-trap dissociation (HCD), the release of the reporter fragments from the TMT-labeled peptide standards provided an internal calibration curve to assess the concentration of these peptides in the CSF. This tool also allowed identifying selectively these peptides in CSF as only the targeted peptides showed specific fragmentation pattern in the TMT reporter-ion zone of the tandem mass spectra. Assays for the concentration measurements of peptides from PARK7, GSTP1, NDKA, and S100B proteins in CSF were further characterized using this novel, efficient, and straightforward approach.

近年来，质谱（mass spectrometry, MS）已成为基于同位素稀释法实现多靶点肽与蛋白质浓度定量的高效分析工具。尽管同量异位标记技术在生物体液潜在生物标志物发现的相对定量领域应用愈发广泛，但目前尚未见其用于绝对定量的实际应用报道。本文采用同量异位串联质量标签（isobaric tandem mass tags, TMTs），对脑脊液（cerebrospinal fluid, CSF）中脑损伤相关蛋白衍生的胰蛋白酶肽进行筛选与定量。合成了蛋白特征性胰蛋白酶肽类似物并配制4种浓度梯度的参考样品，分别采用4种带有m/z=128.1、129.1、130.1及131.1报告离子的同量异位TMT进行差异化标记，并与预先采用TMT 126.1标记的脑脊液样品混合。采用离胶电泳（Off-gel electrophoresis, OGE）作为混合样品的第一维分离手段。所得分离组分采用反相液相色谱（reversed-phase liquid chromatography, RP-LC）串联质谱（tandem mass spectrometry, MS/MS）进行分析，分别使用串联飞行时间（tandem time-of-flight, TOF/TOF）与混合型线性离子阱-轨道阱（hybrid linear ion trap−orbitrap, LTQ−OT）两类质谱仪。在碰撞诱导解离（collision-induced dissociation, CID）或高能C阱解离（higher-energy C-trap dissociation, HCD）条件下，从TMT标记肽标准品上解离得到的报告离子片段可构建内标校准曲线，以此定量脑脊液中对应肽段的浓度。该方法还可实现脑脊液中目标肽的选择性鉴定，原因在于仅靶向肽在串联质谱的TMT报告离子区呈现特异性碎裂模式。采用这一新颖、高效且简便的方法，本研究进一步对脑脊液中PARK7、GSTP1、NDKA及S100B蛋白对应肽段的浓度检测开展了方法学表征。