Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data

Human proteome analysis now requires an understanding of protein isoforms. We recently published the PG Nexus pipeline, which facilitates high confidence validation of exons and splice junctions by integrating genomics and proteomics data. Here we comprehensively explore how RNA-seq transcriptomics data, and proteomic analysis of the same sample, can identify protein isoforms. RNA-seq data from human mesenchymal (hMSC) stem cells were analyzed with our new TranscriptCoder tool to generate a database of protein isoform sequences. MS/MS data from matching hMSC samples were then matched against the TranscriptCoder-derived database, along with Ensembl and the neXtProt database. Querying the TranscriptCoder-derived or Ensembl database could unambiguously identify ∼450 protein isoforms, with isoform-specific proteotypic peptides, including candidate hMSC-specific isoforms for the genes DPYSL2 and FXR1. Where isoform-specific peptides did not exist, groups of nonisoform-specific proteotypic peptides could specifically identify many isoforms. In both the above cases, isoforms will be detectable with targeted MS/MS assays. Unfortunately, our analysis also revealed that some isoforms will be difficult to identify unambiguously as they do not have peptides that are sufficiently distinguishing. We covisualize mRNA isoforms and peptides in a genome browser to illustrate the above situations. Mass spectrometry data is available via ProteomeXchange (PXD001449).人类蛋白质组分析如今亟需对蛋白质异构体（protein isoforms）的深入认知。我们近期发表了PG Nexus分析流程，该流程通过整合基因组学与蛋白质组学数据，可实现外显子（exon）与剪接接头（splice junction）的高可信度验证。本研究全面探究了RNA测序（RNA-seq）转录组学数据与同一样本的蛋白质组分析，可如何用于鉴定蛋白质异构体。我们使用全新的TranscriptCoder工具，对来自人间充质干细胞（human mesenchymal stem cells, hMSC）的RNA-seq数据进行分析，以构建蛋白质异构体序列数据库。随后，将匹配hMSC样本获得的串联质谱（MS/MS）数据，分别与TranscriptCoder构建的数据库、Ensembl数据库及neXtProt数据库进行比对。通过查询TranscriptCoder构建或Ensembl数据库，可明确鉴定约450种蛋白质异构体，这些异构体带有异构体特异性蛋白特征肽（proteotypic peptides），其中包括DPYSL2与FXR1基因的候选hMSC特异性异构体。在缺乏异构体特异性肽段的场景中，非异构体特异性蛋白特征肽组可特异性鉴定多种异构体。上述两类场景中的异构体均可通过靶向串联质谱检测进行鉴定。遗憾的是，本研究分析同时发现，部分异构体因缺乏足够具有区分性的肽段，难以被明确鉴定。我们在基因组浏览器（genome browser）中联合可视化了mRNA异构体与肽段，以阐明上述各类场景。质谱数据可通过ProteomeXchange（PXD001449）获取。