Multiomic, mass spectrometry-based analysis of dried blood for deep phenotyping of sepsis

脓毒症，其特征在于由不受控制的宿主反应引起的危及生命的器官功能障碍， 感染，是医院死亡的主要原因，在美国每年造成> 250，000人死亡。 花费了超过200亿美元的病人护理费用。脓毒症在2年内的死亡率接近50%， 对从严重的COVID-19（一种病毒性败血症）中恢复的患者具有深远的意义。因为败血症是一种 在多器官疾病中，循环蛋白质和代谢物的定量对于脓毒症的分析至关重要 以及其长期影响，并且一些此类测定（乳酸盐、降钙素原）已成为护理标准。 到目前为止，大多数研究，特别是大量的COVID-19脓毒症研究，已经分析了血浆和 与全血相比，血清充满了变异性，掩盖了红细胞的重要作用 和其他细胞类型。为此，杜克大学的生物储存库储存了新冠病毒样本， 重症监护室（ICU）和COVID后门诊诊所的19名脓毒症患者一直在使用 在Neoteryx Mitra尖端上进行体积吸收微量取样（VAMS），以采集和储存全血标本 用于未来的经济分析。该应用程序的首要目标是开发和验证多组学方法 用于Mitra tips中蛋白质和代谢物的基于质谱的定量， 脓毒症病理生物学概况。在R21阶段，我们将开发和验证基于质谱的方法 对于蛋白质和翻译后修饰（磷酸化， 糖基化），并用于多种代谢物类别的靶向定量。我们将开发 实验室间翻译这些方法的参考材料和标准操作程序。在 在R33阶段，我们将分析来自600多名患者时间点的蛋白质、PTM和代谢物， 包含重症脓毒症和COVID-19后队列纵向样本的生物储存库，我们将 将这些数据与精心策划的临床数据集相结合。这些目标的实现将确立血液的效用 由VAMS采样用于未来的脓毒症研究，并将创建一个高度策划的临床和深度多组学数据集 以备将来的假设生成。

Sepsis, which is characterized by life threatening organ dysfunction caused by an uncontrolled host response to infection, is the leading cause of death in hospitals and is responsible for >250,000 deaths per year in the U.S. at the cost of over $20 billion in patient care. Sepsis has a mortality rate nearing 50% at 2 years, which has profound implications for patients recovering from severe COVID-19, a form of viral sepsis. Because sepsis is a multi-organ disease, the quantification of circulating proteins and metabolites is central to the profiling of sepsis and of its long-term effects, and some such assays (lactate, procalcitonin) have become the standard of care. To date, the majority of studies, and particularly the large number in COVID-19 sepsis, have profiled plasma and serum, which, compared to whole blood is fraught with variability and belies the important roles of the erythrocyte and other cell-types. To this end, biorepositories at Duke University, which are banking samples from COVID- 19 sepsis patients in the intensive care unit (ICU) and in a post-COVID outpatient clinic, have been utilizing volumetric absorptive microsampling (VAMS) on Neoteryx Mitra tips to collect and store whole blood specimens for future ‘omic analyses. The overarching goals of this application are to develop and validate multiomic methods for mass spectrometry-based quantification of proteins and metabolites from Mitra tips that comprehensively profile sepsis pathobiology. In the R21 phase, we will develop and validate mass spectrometry-based methods for the non-targeted quantification of proteins and post-translational modifications (phosphorylation, glycosylation) in whole blood, and for targeted quantification of numerous metabolite classes. We will develop reference materials and standard operating procedures for inter-laboratory translation of these approaches. In the R33 phase, we will analyze proteins, PTMs and metabolites from over 600 patient timepoints from biorepositories containing longitudinal samples in critically ill sepsis and post-COVID-19 cohorts, and we will integrate these data with well-curated clinical datasets. Completion of these aims will establish the utility of blood sampling by VAMS for future sepsis studies and will create a highly curated clinical and deep multiomic dataset for future hypothesis generation.