Next Steps on in Silico 2DE Analyses of Chromosome 18 Proteoforms

Abstract

In the boundaries of the chromosome-centric Human Proteome Project (c-HPP) to obtain information about proteoforms coded by chromosome 18, several cell lines (HepG2, glioblastoma, LEH), normal liver, and plasma were analyzed. In our study, we have been using proteoform separation by two-dimensional electrophoresis (2DE) (a sectional analysis) and a semivirtual 2DE with following shotgun mass spectrometry using LC-ESI-MS/MS. Previously, we published a first draft of this research, where only HepG2 cells were tested. Here, we present the next step using more detailed analysis and more samples. Altogether, confident (2 significant sequences minimum) information about proteoforms of 117 isoforms coded by 104 genes of chromosome 18 was obtained. The 3D-graphs showing distribution of different proteoforms from the same gene in the 2D map were generated. Additionally, a semivirtual 2DE approach has allowed for detecting more proteoforms and estimating their pI more precisely. Data are available via ProteomeXchange with identifier PXD010142.

Toward Completion of the Human Proteome Parts List: Progress Uncovering Proteins That Are Missing or Have Unknown Function and Developing Analytical Methods

“…Naryzhny et al 37 report an updated version of their virtual 2DE proteome map based on the analysis of several cell lines (HepG2, glioblastoma, LEH, HEK), normal liver, and plasma, which was coupled to shotgun mass spectrometry using LC− ESI−MS/MS. They claim confident identification of information (minimum of two significant sequences) about the proteoforms of 117 isoforms coded by 104 genes of Chromosome 18, awaiting additional study for validation.…”

Quantitative assessment confirms deep proteome analysis by integrative top–down proteomics

“…Thus, any analysis that genuinely seeks to address the actual breadth and depth of proteomes must therefore have the capacity to routinely yield the necessary high-resolution assessments. Currently, only integrative proteomics-high-resolution front-end proteoform separation by 2DE and subsequent detailed spot dissection by LC/MS/MS-provides such necessary rigor [3,4,24].…”

“…Thus, any analysis that genuinely seeks to address the actual breadth and depth of proteomes must therefore have the capacity to routinely yield the necessary high‐resolution assessments. Currently, only integrative proteomics—high‐resolution front–end proteoform separation by 2DE and subsequent detailed spot dissection by LC/MS/MS—provides such necessary rigor [3, 4, 24]. In our ongoing efforts to quantitatively test and refine integrative top–down proteomic analyses, here we have evaluated possible proteoform losses during facedown and faceup IPG strip rehydration and the IEF steps of 2DE.…”

Puzzle of Proteoform Variety—Where Is a Key?

“…After specific hydrolysis, further analyses can be performed by the bottom-up MS [ 21 , 22 ]. Actually, based on 2DE separation followed by bottom-up MS, the proteoform profiles were generated for several types of cells [ 23 , 24 , 25 , 26 ]. What is more, these data were used to generate a web database called “2DE-pattern” [ 27 ].…”