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（Endothelial cells, ECs）是所有血管的腔面内衬，对心血管系统的正常运作至关重要。其表型可通过RNA可变剪接进行调控，进而产生不同的蛋白质异构体（protein isoform）。为了刻画内皮细胞内的RNA与蛋白质异构体图谱，本研究采用长读长蛋白质基因组学（long read proteogenomics）方法，对人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）进行分析。由PacBio测序得到的转录本作为样本特异性蛋白质数据库的构建基础，该数据库用于后续的质谱（mass-spectrometry, MS）分析，以推断蛋白质异构体的表达情况。本研究从10426个基因中鉴定出53863个转录本异构体（transcript isoform），其中22195个为全新转录本。此外，人脐静脉内皮细胞中占主导的异构体有25%并不符合公认的参考异构体（reference isoform），血管通路相关基因便属于这一类群。本研究鉴定出2597个可通过唯一肽段验证的蛋白质异构体，结合长读长转录本证据后，又额外鉴定出2280个蛋白质异构体。我们还对内皮相关基因CDH5的全新可变剪接受体位点进行了表征，这提示其相关信号通路可能发生改变。最终，我们通过唯一比对的全新肽段，鉴定出由多种RNA剪接机制产生的新型蛋白质异构体。本研究的结果构建了一张高分辨率的已知与新型异构体图谱，其或与内皮细胞表型及功能密切相关。