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.

Haptoglobin (Hp) is a blood plasma glycoprotein that binds free hemoglobin (Hb) and plays a critical role in tissue protection and the prevention of oxidative damage. In addition, it has a number of regulatory functions. Haptoglobin is an acute phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein synthesized mainly in the liver and lungs and is the subject of research as a potential biomarker of many diseases, including various forms of malignant neoplasms. Haptoglobin has several unique biophysical characteristics. Only in humans, the Hp gene is polymorphic, has three structural alleles that control the synthesis of three major phenotypes of Hp, homozygous Hp1-1 and Hp2-2, and heterozygous Hp2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual's predisposition to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). These are structural transformations (removal of the signal peptide, cutting of the Pre-Hp precursor molecule into two subunits, α and β, limited proteolysis of α-chains, formation of disulfide bonds, multimerization), as well as chemical modifications of α-chains and glycosylation of the β-chain. Glycosylation of the β-chain of haptoglobin at four Asn sites is the most important variable PTM that regulates the structure and function of the glycoprotein. The study of modified oligosaccharides of the Hp β-chain has become the main direction in the study of pathological processes, including malignant neoplasms. Many studies are focused on the identification of PTM and changes in the level of the α2-chain of this protein in pathology. These characteristics of Hp indicate the possibility of the existence of this protein as different proteoforms, probably with different functions. This review is devoted to the description of the structural and functional diversity of Hp and its potential use as a biomarker of various pathologies.

Haptoglobin (Hp) is a blood plasma glycoprotein that plays a critical role in tissue protection and the prevention of oxidative damage. Haptoglobin is an acute-phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein and is the subject of research as a potential biomarker of many diseases, including malignant neoplasms. The Human Hp gene is polymorphic and controls the synthesis of three major phenotypes—homozygous Hp1-1 and Hp2-2, and heterozygous Hp2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual’s predisposition to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). Glioblastoma multiform (GBM) is the most malignant primary brain tumor. In our study, we have analyzed the state of Hp proteoforms in plasma and cells using 1D (SDS-PAGE) and 2D electrophoresis (2DE) with the following mass spectrometry (LC ES-MS/MS) or Western blotting. We found that the levels of α2- and β-chain proteoforms are up-regulated in the plasma of GBM patients. An unprocessed form of Hp2-2 (PreHp2-2, zonulin) with unusual biophysical parameters (pI/Mw) was also detected in the plasma of GBM patients and glioblastoma cells. Altogether, this data shows the possibility to use proteoforms of haptoglobin as a potential GBM-specific plasma biomarker.

— Haptoglobin (Hp) is a glycoprotein that binds free hemoglobin (Hb) in plasma and plays a critical role in tissue protection and prevention of oxidative damage. Besides, it has some regulatory functions. Haptoglobin is an acute-phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein synthesized mainly in the liver and lungs and is the subject of research as a potential biomarker of many diseases, including various forms of malignant neoplasms. Haptoglobin has several unique biophysical characteristics. The human Нр gene is polymorphic, has three structural alleles that control the synthesis of three major phenotypes of haptoglobin: homozygous Нр1-1 and Нр2-2, and heterozygous Нр2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual predisposition of a person to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). These are structural transformations (removal of the signal peptide, cutting off the Pre-Hp precursor molecule into two subunits, α and β, limited proteolysis of α-chains, formation of disulfide bonds, multimerization), as well as chemical modifications of α-chains and glycosylation of the β-chain. Glycosylation of the β-chain of haptoglobin at four Asn sites is the most important variable PTM that regulates the structure and function of the glycoprotein. The study of modified oligosaccharides of the β-chain of Hp has become the main direction in the study of pathological processes, including malignant neoplasms. These characteristics indicate the possibility of the existence of Hp in the form of a multitude of proteoforms, probably performing different functions. This review is devoted to the description of the structural and functional diversity and the potential use of Hp as a biomarker of various pathologies.

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.