Proteomic Signature of Endothelial Dysfunction Identified in the Serum of Acute Ischemic Stroke Patients by the iTRAQ-Based LC–MS Approach

Acute
ischemic stroke (AIS) is a devastating cerebrovascular disorder
that leads to permanent physical and neurological disabilities in
adults worldwide. Proteins associated with stroke pathogenesis may
appear in the serum of AIS patients due to blood–brain barrier
dysfunction, thus permitting the development of blood-based biomarkers
for early diagnosis of stroke. These biomarkers could perhaps be an
adjunct to the existing imaging modalities and aid in better management
and therapeutic intervention during the course of the disease. For
this exploratory study, a combination of multiplexed isobaric tagging
using iTRAQ reagents and high resolution tandem mass spectrometry
was used to identify differentially expressed proteins in serum samples
from AIS patients. The quantitative proteomic analysis of serum from
both AIS and control subjects revealed 389 high confidence protein
identifications and their relative levels. Among them, 60 proteins
showed a ≥1.5-fold change in the AIS subjects. We verified
the altered serum levels of candidate proteins such as vWF, ADAMTS13,
S100A7, and DLG4 through ELISA, and the results also corroborate with
the experimental findings. vWF and ADAMTS13 are key players that regulate
blood hemostasis, and their altered concentration may contribute to
endothelial dysfunction. S100A7 is a novel candidate protein identified in this
study that is also known to mediate inflammation, endothelial proliferation,
and angiogenesis. The current study provided a potential and novel
biomarker panel that may in turn provide diagnostic aid to the existing
imaging modalities for the rapid diagnosis of ischemic stroke

急性缺血性脑卒中（Acute Ischemic Stroke, AIS）是一种极具破坏性的脑血管疾病，会导致全球范围内成年患者出现永久性躯体及神经功能残疾。由于血脑屏障功能受损，与脑卒中发病机制相关的蛋白质可出现在AIS患者的血清中，这为开发基于血液的脑卒中早期诊断生物标志物提供了可能。此类生物标志物可作为现有影像学检测手段的辅助工具，助力疾病进程中的精细化管理与治疗干预。本探索性研究采用同位素相对与绝对定量（isobaric Tags for Relative and Absolute Quantitation, iTRAQ）试剂进行多重等压标记结合高分辨串联质谱的联用策略，对AIS患者血清样本中的差异表达蛋白质进行鉴定。对AIS患者与健康对照者的血清进行定量蛋白质组学分析后，共鉴定出389个高可信度蛋白质，并获得其相对表达水平。其中，60种蛋白质在AIS患者群体中呈现≥1.5倍的表达差异。本研究通过酶联免疫吸附实验（Enzyme-Linked Immunosorbent Assay, ELISA）验证了血管性血友病因子（von Willebrand Factor, vWF）、ADAM金属肽酶域13（ADAM Metallopeptidase with Thrombospondin Type 1 Motif 13, ADAMTS13）、S100A7及DLG4等候选蛋白质的血清水平异常，结果与前述实验发现相吻合。vWF与ADAMTS13是调控血液止血过程的关键因子，其浓度异常可能与内皮功能障碍相关。S100A7是本研究鉴定出的新型候选蛋白质，已知其可介导炎症反应、内皮细胞增殖与血管生成过程。本研究构建了一套具有应用潜力的新型生物标志物组合，可为现有影像学检测手段提供辅助，助力缺血性脑卒中的快速诊断。