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

Abstract

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.

Towards the Full Realization of 2DE Power

“…Also, taking into account recent publications [88,89], we can reconsider the major steps in 2DE-based proteomics. In the near future, they would be

sample preparation/extraction

protein separation by 2DE

protein gel staining (Coomassie)

proteoform location, identification, characterization, and quantitation by ESI LC-MS/MS

virtual/experimental 2DE protein database construction

…”

“…Only the proteins detected as spots after staining are cut out and analyzed. This dilemma can be solved only by analyzing all parts of the gel [89,90]. Using this approach, we are not losing any proteoform that was separated by 2DE and present inside the gel.…”

“…Accordingly, the same proteins detected in different sections were considered as different proteoforms. About 20,000 proteoforms coded by ~4000 genes were identified using this approach [89,90]. Moreover, information about the set of proteoforms coded by each of these genes can be extracted and represented separately as a 3D chart (Figure 2).…”

“…Another problem is proteoform quantitation. A quantitation parameter often used, the exponentially modified form of protein abundance index (emPAI) [91] used in the papers [89,90] is not ideal, since it gives only a relative and not very accurate estimation of protein content. Hopefully, the situation here will be improved, keeping in mind the rapid progress in mass spectrometry.…”

“…Here, the combination of 2DE with ESI LC-MS/MS looks like a good choice, since together they allow better representation of the information obtained [88,89]. In one example [88], proteins (cell extract and human blood plasma) were run by 2DE.…”