Plasma proteomics in two Swedish cohorts with Ovarian Cancer

The plasma proteome was analyzed using the proximity extension assay (PEA) as implemented in the Explore HT-version (Olink Proteomics AB, Uppsala, Sweden). The samples were analyzed at the Olink Service Laboratory in Uppsala, Sweden. In brief, the PEA is based on pairs of antibodies equipped with probes, DNA single-strand oligonucleotide reporter molecules, that bind to their respective target if present. Target binding by both probes in close proximity generates double-stranded DNA amplicons which are then quantified by next generation sequencing. Here, 5416 unique proteins were characterized in each of the 404 samples. The samples were randomized across plates with respect to cohort and diagnose (benign or malignant). In the resulting data-file provided by the analysis platform, each individual protein measurement, assay and sample is labelled depending on passing or failing quality control as provided by the instrument software. Here, a total on 768 individual measurements (corresponding to 0.036%, 768/(404*5416)) did not pass quality control and were removed from further analyses. Two proteins (apolipoprotein E (APOE) and fibronektin 1 (FN1)) had detection rates below 95% across all samples and were removed from further analyses. The resulting NPX values are on a log2 scale and in the logarithmic phase of the curve, one (1) increase of the NPX value corresponds to a doubling of the protein content. In the resulting data, a high NPX value corresponds to a high protein concentration. Each of the measured proteins has a lower limit of detection (LOD) given in the same NPX-scale which is determined at run time. Here, measurements under LOD were kept as is in the downstream analysis. After quality control, the detection rate across all samples were 99.5 to 99.8%.本研究采用邻位延伸分析（proximity extension assay, PEA）的Explore HT版本（瑞典乌普萨拉Olink Proteomics AB公司提供）对血浆蛋白质组进行分析。所有样本均在瑞典乌普萨拉的Olink服务实验室完成检测。 简而言之，邻位延伸分析基于搭载单链DNA寡核苷酸报告分子探针的抗体对：若靶标蛋白存在，探针可与其特异性结合。当两枚探针紧密结合至同一靶标时，即可生成双链DNA扩增子，随后通过下一代测序（next generation sequencing）实现定量。 本研究共纳入404份样本，每份样本均完成5416种独特蛋白质的表征分析。所有样本按照队列与诊断类型（良性或恶性）进行板间随机化处理。 在分析平台输出的原始数据文件中，每一项蛋白质检测结果、检测项目及样本均会根据仪器软件提供的质量控制（quality control, QC）标准标记为合格或不合格。本研究中，总计768项单独检测结果（占比0.036%，即768/(404×5416)）未通过质量控制，予以剔除。另有两种蛋白质——载脂蛋白E（apolipoprotein E, APOE）与纤连蛋白1（fibronektin 1, FN1）——在所有样本中的检出率低于95%，同样被排除在后续分析之外。 本研究得到的NPX值以log₂尺度呈现，处于曲线的对数线性区间内：NPX值每增加1，对应蛋白质含量翻倍；NPX值越高，则代表对应的蛋白质浓度越高。每一种被检测的蛋白质均配有同一NPX尺度下的检测下限（limit of detection, LOD），该下限由检测运行过程中确定。本研究中，低于检测下限的检测结果在下游分析中予以保留。 经质量控制处理后，所有样本的整体检出率为99.5%至99.8%。