Translating Evidence-aided Proteome Analysis: A Strategic Demonstration

Chromosome-centric human proteome project(C-HPP) aims at differentiatingchromosome-based and tissue-specificprotein compositions in terms of proteinexpression,quantification, and modification. We previously found that theanalysis of translatingmRNA (mRNA attached to ribosome-nascentchain complex, RNC-mRNA) can explain over94%of mRNA-protein abundance. Therefore, we propose here to usefull-length RNC-mRNAinformation to illustrate proteinexpression both qualitatively and quantitatively.Aim:Demonstrationthe potential and necessityof including translating mRNA analysis as thefourth pillar of C-HPP.Methods:We extracted RNC-mRNA (RNC-seq)and total mRNA from human normal bronchialepithelial(HBE) cells and human colorectal adenocarcinoma Caco-2cells, followed by nextgeneration sequencing and RNC-mRNA analyses. We examined the profiling and sequencecoverage characteristics of RNA-mRNA sequencing. In Caco-2cells, we aligned and assayed thetranslating evidence with currently available proteome data. In addition, the translationefficiency of single-nucleotide variations was determined. Furthermore, we analyzeddifferentially-translated genes and cell-specific translating genes comparing the two cell lines ina chromosome-by-chromosome manner..Results:We detected12,758and14,113translating genes in HBE cells and Caco-2cells,respectively. And found that most of them were mappedwith>80%of coding sequencecoverage. In Caco-2cells, we provided translating evidence on4,180significantsingle-nucleotidevariations. While using RNC-mRNA data as a standard for proteomic dataintegration, both translating and protein evidence of7,876genes can be acquired from fourinter-laboratory data sets with different MS platforms, suggesting that there are approximately6,000translating genes, which have no mass spectrometry evidence for Caco-2cells.To be noted,we detected1,397noncodingmRNAs that were attached to ribosomes, suggesting a potentialsource of new protein explorations. By comparing the two cell lines, atotal of677differentiallytranslated genes were found to be non-evenly distributed across chromosomes. Furthermore, we identified that2,105genes inCaco-2and750genes in HBE cells wereexpressed in a cell-specificmanner. These genes weresignificantly and specifically clustered onspecific chromosomes, suchas chromosome19.Conclusions:We proposed that the HPP/C-HPP goal of finding missing and new proteins can beimproved byintegrating RNC-mRNA analysis in addition to MS, bioinformatics, andantibody-based verifications.