J Proteome Res. 2016 Feb 5;15(2):525-30. doi: 10.1021/acs.jproteome.5b00871.
Virtual-Experimental 2DE Approach in Chromosome-Centric Human Proteome Project.
Naryzhny SN1,2, Maynskova MA1, Zgoda VG1, Ronzhina NL2, Kleyst OA2, Vakhrushev IV1, Archakov AI1.
- 1Institute of Biomedical Chemistry of Russian Academy of Medical Sciences , Pogodinskaya 10, Moscow 119121, Russia.
- 2Petersburg Nuclear Physics Institute, National Research Center “Kurchatov Institute” , Leningrad Region, Gatchina 188300, Russia.
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 41 genes of 18th chromosome. The 3D graphs showing the distribution of different proteoforms from the same gene in 2D map were generated.
KEYWORDS: Chromosome 18; ESI LC−MS/MS; chromosome-centric; inventory; mass spectrometry; proteoforms; proteome; two-dimensional electrophoresis
The final goal of proteomics is complete information about all proteins. It’s evident that this goal is not reachable in near future, even despite of modern bust of proteomics. For better international cooperation in proteomics, “Human Proteome Project” (HPP) was officially launched at 9th HUPO congress (2010) in Sydney, Australia. HPP was initiated to create a comprehensive knowledge base of all human proteins for using as a tool in different areas like education, science, or medicine that need this information. It was agreed that the project will be chromosome-centric (C-HPP) to promote more effective collaborations1-3. Russia decided to be responsible for Chromosome 18 that harbors 492 annotated genes including 276 protein coding genes. As these proteins are presented as multiple variants/proteoforms/protein species, we expect to detect at least 3000 proteoforms generated from Chromosome 18. There is a long list of diseases related to proteins coded by Chromosome 18 (psoriasis, Parkinson’s disease, Alzheimer’s disease, arthritis, osteosarcoma, colorectal cancer…). Some of proteins connected to these diseases are: serpins, Netrin receptor DCC precursor, Niemann-Pick C1 protein precursor , the regulator of apoptosis BCL2, the members of SMAD family. Our project involves bioinformatics and experimental approaches – search and selection of comprehensive information about all proteins coded by genes of chromosome 18, starting from transcriptome, calculation, quantitative and qualitative analysis of different proteoforms in different types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma; using mass-spectrometry of high sensitivity and resolution4-7. Development of interactive virtual 2DE map of proteins coded by genes of chromosome 18 in combination with experimental 2DE protein map will allow executing more effectively the Russian part of C-HPP, to estimate more accurately number of proteoforms, and will be a basis for the knowledgebase of human proteins. As protein extracts of cancer cells are used in our study, information about proteoform distribution (proteome) will allow better understanding of protein dynamic in cancerogenesis, bearing in mind that our next step will be the similar analysis of normal cells (hepatocytes). It will allow the identification of characteristic quantitative and qualitative differences between normal and cancer cells for use as biomarkers or targets for therapy. Our calculations showed that there are at least 70 thousand different protein proteoforms in a single differentiated human cell8. Further analysis (identification and quantitative estimation) will allow calculating number of all proteoforms and giving us a clue of their distribution and functioning inside a cell. Here, we represent our first step in this direction based on HepG2 cells. Further study will involve human liver, plasma and other types of cells (normal and cancer). In our study, we collected information about known proteoforms coded by genes of 18th human chromosome. At first the theoretical pI and Mw of these proteoforms were calculated and possible position on HepG2 2DE picture was estimated (Figure1A). Next, the gel was divided into 96 sections, identified as 1- 12 along the Mw dimension and A – H along the pI dimension (Figure 1B). All these gel sections were cut and treated with trypsin according to protocol for mass-spectrometry by LC-ESI-MS/MS. The tryptic peptides obtained from each 2DE section were analyzed using an Orbitrap Q-Exactive mass spectrometer and Mascot “2.4.1”. All proteins detected in the same section were given the pI/Mw parameters of this section. For instance, a section B4 has coordinates: pI 4.45-5.11 / Mw 52000-64000 and a section B6 – pI 4.45-5.11 / Mw 35000-40000 (Figure 2B). If the same protein (product of the same gene) was identified in different sections it was considered to exist in different proteoforms. After analysis of all sections and protein identification, a redundant list of 20462 protein names was generated. This list contains 3774 unique gene names. Among them, 165 proteoforms are representing 41 genes of 18th chromosome. The 3D-graphs showing distribution of proteoforms coded by the same gene in 2DE map were generated. Some of them are shown in Figures 3. Analysis of data presented in Figures 3 gives us a general view about a single gene proteomes (products of the same gene). According to this analysis, a master protein usually was presented as a most abundant proteoform in these graphs. With this study, we presented for the first time an approach of combination of theoretical information about known proteoforms (master forms and splice variants) coded by chromosome 18 based on 2DE with experimental data obtained by 2DE and LC ESI-MS/MS. This is a first step in creation of 2DE-based knowledge database of proteins coded by 18th chromosome.
The importance of this study
We produce a large volume of information about multiple proteoforms coded not only by chromosome 18 but other chromosomes as well. Further analysis of these proteoforms will give a key for study and cure of many diseases including cancer9,10.
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This work was funded by a grant of RSF (Russian Science Foundation ) # 14-25-00132 from 15.08.2014 (RSF # 14-25-00132).
Contact: Dr. Stanislav N. Naryzhny,
Institute of Biomedical Chemistry of Russian Academy of Medical Sciences, Pogodinskaya 10, Moscow, 119121, Russia
Petersburg Nuclear Physics Institute, Gatchina, Leningrad district, 188300, Russia.
Tel: (+7) 9111764453. E-mail: email@example.com