Identification and quantitative analysis of different proteoforms (protein species) presented in a cell line generated from high grade glioblastoma was performed using two-dimensional electrophoresis (2DE), mass spectrometry (ESI LC-MS/MS), and immunodetection. A 2DE protein map containing an extensive data set comprising 937 spots with 1542 unique protein identifications (proteoforms) of 600 genes was obtained. Additionally, another set of experiments was performed where 16012 proteoforms coded by 4050 genes were identified by MS/MS according to their position in 96 gel sections (pixels). A special attention has been paid to the proteins that are the potential biomarkers of glioblastoma. The list of these biomarkers was compiled from literature. Next, we generated the graphs with theoretical and experimental information about proteoforms coded by the same gene. Such a virtualexperimental representation allowed better visualization of the state of these gene products. Many proteins, potential biomarkers of glioblastoma as well, are characterized by high numbers of protein species. We assume that these species could be a potential source of highly specific biomarkers of glioblastoma.

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The human proteome is composed of a diverse and heterogeneous range of gene products/proteoforms/protein species. Because of the growing amount of information about proteoforms generated by different methods, we need a convenient approach to make an inventory of the data. Here, we present a database of proteoforms that is based on information obtained by separation of proteoforms using 2DE followed by shotgun ESI–LC–MS/MS. The database's principles and structure are described. The database is called “2DE‐pattern” as it contains multiple isoform‐centric patterns of proteoforms separated according to 2DE principles. The database can be freely used at http://2de-pattern.pnpi.nrcki.ru.

Mammalian nuclear DNA polymerases a and p are known to be devoid of the editing 3'-+5' exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'+5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-50) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A-T)] template, respectively, 10-fold and 2-fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase a from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage 6x174 amber 3 is increased 5-lO-fold, levels of exonuclease and DNApolymerase activities being similar. Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the a-polymerase suggests that exonucleolytic proofreading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.Fidelity of DNA synthesis depends on the sum of at least two processes : the accuracy of matched nucleotide selection effected by DNA polymerase, and the 3'45' exonucleolytic correction of possible misinsertions. Incorrect bases incorporated during replication and not corrected by a proof-reading function may be recognized by a post-replicative mismatchrepair system which is yet to be characterized biochemically and will not be considered here. Proof-reading enhances the accuracy of DNA synthesis by 2-3 orders in the case of prokaryotic DNA polymerases with 3'+5' exonuclease activity [l]. However, highly purified mammalian nuclear DNA polymerases a and p lack associated 3'+5' exonuclease activity while polymerase 6 and E display rather low levels of this activity, and their accuracy is only 1-2 orders higher than that of DNA polymerase a [1, 21. In addition, the biological ubiquity of DNA polymerases 6 and E is still doubtful. Exonucleases non-covalently associated with DNA polymerases were also suggested to proof-read in mammalian nuclei 131. Eligible 3'45' exonucleases were found in rabbit bone marrow [4], calf spleen 131, rat liver [5, 61, rat hepatoma [7], and human cells [S, 91. However, the real influence of these exonucleases on the fidelity of synthesis with DNA polymerase has not been investigated. Whether such exonucleases play any part in the overall fidelity of mammalian DNA replication is still a matter of debate.

Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.