ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing
ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing
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DOI：
10.1371/journal.pone.0025583
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发表时间：
2011-09-29
期刊：
影响因子：
3.7
通讯作者：
Landegren, Ulf
中科院分区：
文献类型：
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作者：
Darmanis, Spyros;Nong, Rachel Yuan;Landegren, Ulf
尽管有强烈的兴趣，但在许多样品中提供增强敏感性和特异性的候选蛋白质生物标志物平行测量方法一直缺乏。我们在此提出了一种多重接近连接试验，通过实时PCR或DNA测序（ProteinSeq）进行读数。我们证明了在同时分析5 μ l血浆中蛋白质组时，比传统的夹心测定法提高了灵敏度。重要的是，我们观察到与夹心实验相比，多路复用的最小趋势增加了背景，这表明更高水平的多路复用是可能的。我们使用ProteinSeq分析来自心血管疾病（CVD）患者队列和匹配对照的血浆样本中的蛋白质。三种蛋白，即p -选择素，Cystatin-B和Kallikrein-6被确定为CVD的推定诊断生物标志物。后两者在以前的文献中没有报道过，它们的潜在作用必须在更大的患者队列中得到验证。我们得出的结论是，ProteinSeq在筛选大量蛋白质和样品方面很有前景，同时该技术可以为生物样本库样本和临床使用的诊断标记物的验证提供急需的平台。
Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of proteins in 5 mu l of blood plasma. Importantly, we observe a minimal tendency increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6 were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising or screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biabank samples and in clinical use.