Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells

Abstract

The final goal of the Russian part of the Chromosome-centric Human Proteome Project (C-HPP) was established as the analysis of the chromosome 18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells with the sensitivity of 10(-18) M. Using SRM, we have recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2 cells. On the basis of the results of the survey, the SRM assays were drafted for 250 proteins: 41 proteins were found only in the liver tissue, 82 proteins were specifically detected in depleted plasma, and 127 proteins were mapped in both samples. The targeted analysis of HepG2 cells was carried out for 49 proteins; 41 of them were successfully registered using ordinary SRM and 5 additional proteins were registered using a combination of irreversible binding of proteins on CN-Br Sepharose 4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq and RT-PCR has shown a significant correlation (r = 0.78) for 42 gene transcripts. A pilot affinity-based interactome analysis was performed for cytochrome b5 using analytical and preparative optical biosensor fishing followed by MS analysis of the fished proteins. All of the data on the proteome complement of the Chr 18 have been integrated into our gene-centric knowledgebase ( www.kb18.ru ).

Dataset of target mass spectromic proteome profiling for human chromosome 18

“…List of peptides for 268 chromosome 18 proteins was generated manually using data about occurrence of proteotypic peptides from proteomic repositories GPMdb, ProteinAtlas and PRIDE and MaRiMba-criteria (protocol was described earlier in Supplementary note 2, Zgoda et al [1]). For each protein one “best-flyer” peptide was chosen.…”

“…LC-SRM Analysis was held as described earlier in Supplementary note 2, Zgoda et al [1]. Each SRM experiment was repeated in 3 technical runs.…”

“…Proteome profiling was performed using stable isotope-labeled standards (SRM/SIS) for plasma, liver tissue and HepG2 cells. Dataset includes both positive and negative results of protein detection.These data were partly discussed in recent publications, “Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells” [1] and “Chromosome 18 transcriptoproteome of liver tissue and HepG2 Cells and targeted proteome mapping in depleted plasma: Update 2013” [2], supporting the accompanying publication “State of the Chromosome 18-centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells” [3], and are deposited at the ProteomeXchange via the PASSEL repository with the dataset identifier PASSEL: PASS00697 for liver and HepG2 cell line.…”

Recent advances in proteomic profiling of human blood: clinical scope

“…As a result, SRM traces for 265 proteins were collected, among them 164 proteins were identified and measured with high confidence (for details see [18]). Distribution of the number of copies per 1 ml of depleted plasma for Chr 18-encoded proteins [6,7] is of asymmetric bell-shaped character with the modal copy number of 10 8 copies. For comparison, application of intensive shotgun-MS made it possible to identify only 55 proteins (~20% of the proteins of Chr 18) [19].…”

“…Application of the proteome chromosome-centric approach [5] in combination with selected reaction monitoringmass spectrometry (SRM-MS) made it possible to raise the study of plasma proteome to a new level: with the use of this method, the results of the qualitative and quantitative proteome measurements are mapped onto protein coding genes of the chromosomes [6,7].…”