Human peripheral blood mononuclear cells (PBMC) are key to several diagnostics assays and basic science research. Blood pre-analytical variations that occur before obtaining the PBMC fraction can significantly impact the assays results, including viability, composition, integrity, and gene expression changes of immune cells. With this as motivation, we performed a quantitative shotgun proteomics analysis using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ 8plex) labeling to compare PBMC obtained from 24 h-stored blood at room temperature versus freshly isolated. We identified a total of 3195 proteins, of which 245 were differentially abundant (101 upregulated and 144 downregulated). Our results revealed enriched pathways of downregulated proteins related to exocytosis, localization, vesicle-mediated transport, cell activation, and secretion. In contrast, pathways related to exocytosis, neutrophil degranulation and activation, granulocyte activation, leukocyte degranulation, and myeloid leukocyte activation involved in immune response were enriched in upregulated proteins, which may indicate probable granulocyte contamination and activation due to blood storage time and temperature. Examples of upregulated proteins in the 24 h-PBMC samples are CAMP, S100A8, LTA4H, RASAL3, and S100A6, which are involved in an adaptive immune system and antimicrobial activity, proinflammatory mediation, aminopeptidase activities, and naïve T cells survival. Moreover, examples of downregulated proteins are NDUFA5, TAGLN2, H3C1, TUBA8, and CCT2 that are related to the cytoskeleton, cell junction, mitochondrial respiratory chain. In conclusion, the delay in blood-processing time directly impacts the proteomic profile of human PBMC, possibly through granulocyte contamination and activation.

人外周血单核细胞（PBMC）是几种诊断测定和基础科学研究的关键。在获得PBMC馏分之前发生的血液分析前变化会显着影响测定结果，包括免疫细胞的活力，组成，完整性和基因表达变化。以此为动力，我们使用等压相对和绝对定量标签（iTRAQ 8plex）进行了散弹枪蛋白质组学分析，比较了室温和新鲜分离的24 h血液中的PBMC。我们鉴定出总共3195种蛋白质，其中245种差异丰富（上调101种，下调144种）。我们的研究结果揭示了与胞吐作用，定位，囊泡介导的转运，细胞活化，和分泌物。相反，参与免疫应答的与胞吐作用，嗜中性粒细胞脱粒和活化，粒细胞活化，白细胞脱粒和髓样白细胞活化相关的途径富含上调的蛋白质，这可能表明由于血液储存时间和温度而可能导致粒细胞污染和活化。在24 h-PBMC样品中上调的蛋白质的例子是CAMP，S100A8，LTA4H，RASAL3和S100A6，它们参与了适应性免疫系统和抗菌活性，促炎介质，氨肽酶活性和幼稚T细胞存活。此外，下调蛋白质的例子是与细胞骨架，细胞连接，线粒体呼吸链有关的NDUFA5，TAGLN2，H3C1，TUBA8和CCT2。结论，