Virtual-Experimental 2DE Approach in Chromosome-Centric Human Proteome Project.

To obtain more information about human proteome, especially about proteoforms (protein species) coded by 18th chromosome, we separated proteins from human cancer cell line (HepG2) by two-dimensional gel electrophoresis (2DE). Initially, proteins in major spots were identified by MALDI-MS peptide mass fingerprinting. According to parameters (pI/Mw) of identified proteins the gel was calibrated. Using this calibrated gel, a virtual 2D map of proteoforms coded by Chromosome 18 was constructed. Next, the produced gel was divided into 96 sections with determined coordinates. Each section was cut, shredded, and treated by trypsin according to mass-spectrometry protocol. After protein identification by shotgun mass spectrometry using ESI LC-MS/MS, a list of 20 462 proteoforms (product of 3774 genes) was generated. Among them, 165 proteoforms are representing 39 genes of 18th chromosome. The 3D graphs showing the distribution of different proteoforms from the same gene in 2D map were generated. This is a first step in creation of 2DE-based knowledge database of proteins coded by 18th chromosome.