Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted “reference isoform” 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.

内皮细胞（ECs）构成了所有血管的内腔衬里，对于心血管系统的功能至关重要。其表型可通过RNA的可变剪接来调节，从而产生不同的蛋白质异构体。为了表征内皮细胞内RNA和蛋白质异构体的图谱，我们采用长读长蛋白质组学方法分析了人类脐静脉内皮细胞（HUVECs）。从PacBio测序中得到的转录本构成了特定样本的蛋白质数据库的基础，用于下游质谱（MS）分析，以推断蛋白质异构体的表达。我们检测到来自10,426个基因的53,863个转录本异构体，其中22,195个转录本为新型。此外，在HUVECs中，主导的异构体有25%的时间并不对应于公认的“参考异构体”，其中血管通路相关基因亦包含在内。我们发现了2,597个由独特肽段支持的蛋白质异构体，通过纳入长读长转录本证据，还确定了额外的2,280个异构体。我们表征了内皮相关基因CDH5的一个新型可接受位点，这暗示了其相关信号通路可能发生的改变。最终，我们识别出由多种RNA剪接机制产生的、由独特映射的新型肽段支持的蛋白质新异构体。我们的研究结果代表了一个高分辨率的图谱，其中包含了与内皮细胞表型和功能相关的已知和新型异构体。