Biomedical subjects

T A Nyman

Publications and source records attributed to T A Nyman.

7 recordsLinked to original sources

PubMed2005

Geometrical distortions in two-dimensional gels: applicable correction methods.

Two-dimensional electrophoresis (2-DE) provides a rapid means for separating thousands of proteins from cell and tissue samples in one run. Although this powerful research tool has been enthusiastically applied in many fields of biomedical research, accurate analysis and interpretation of the data have provided many challenges. Several analysis steps are needed to convert the large amount of noisy data obtained with 2-DE into reliable and interpretable biological information. The goals of such analysis steps include accurate protein detection and quantification, as well as the identification of differentially expressed proteins between samples run on different gels. To achieve these goals, systematic errors such as geometric distortions between the gels must be corrected by using computer-assisted methods. A wide range of computer software has been developed, but no general consensus exists as standard for 2-DE data analysis protocol. The choice of analysis approach is an important element depending both on the data and on the goals of the experiment. Therefore, basic understanding of the algorithms behind the software is required for optimal results. This review highlights some of the common themes in 2-DE data analysis, including protein spot detection and geometric image warping using both spot- and pixel-based approaches. Several computational strategies are overviewed and their relative merits and potential pitfalls discussed. Finally, we offer our own personal view of future trends and developments in large-scale proteome research.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2001

A proteome database of human primary T helper cells.

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.

Databases, Protein↗

PubMed2001

The role of mass spectrometry in proteome studies.

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.

Databases, Protein↗

PubMed2001

Scots pine expresses short-root-specific peroxidases during development.

Nine short-root-specific proteins from Scots pine (Pinus sylvestris L.) detected and isolated as individual spots by 2D-PAGE were identified. The similar peptide mass maps obtained for all nine polypeptide spots together with lectin-blotting results suggest that they represent forms of the same modified protein. N-Terminal sequence analysis of two of the peptides showed high similarity to peroxidases. RT-PCR with oligonucleotide primers corresponding to determined peptide sequences and conserved regions in plant peroxidases led to isolation of Psyp1 cDNA which is most abundantly expressed in short roots. Psyp1 is the first peroxidase cDNA to be isolated from the genus Pinus. It encodes a 363-amino-acid class-III peroxidase with a calculated molecular mass of 35.7 kDa and theoretical pI of 4.74. The predicted PSYP1 amino-acid sequence is grouped with other class-III peroxidases in phylogenetic analyses, but it has a unique amino-acid sequence which may be associated with its function in short roots or with its phylogenetic group. The presence of a signal sequence for extracellular transport indicates that PSYP1 belongs to the group of secreted class-III peroxidases. The presence of 10 tyrosine residues and putative auxin-binding regions in PSYP1 suggests that the function of the enzyme is associated with cell-wall formation in short roots. The downregulation of Psyp1 expression in symbiotic short roots hosting the ectomycorrhizal fungus Suillus bovinus is perhaps related to the change in cell-wall structure necessary for ectomycorrhizal development.

Amino Acid Sequence↗

PubMed2000

Proteome analysis reveals ubiquitin-conjugating enzymes to be a new family of interferon-alpha-regulated genes.

Interferon (IFN)-alpha is a cytokine with antiviral, antiproliferative, and immunomodulatory properties, the functions of which are mediated via IFN-induced protein products. We used metabolic labeling and two-dimensional gel electrophoresis followed by MS and database searches to identify potentially new IFN-alpha-induced proteins in human T cells. By this analysis, we showed that IFN-alpha induces the expression of ubiquitin cross-reactive protein (ISG15) and two ubiquitin-conjugating enzymes, UbcH5 and UbcH8. Northern-blot analysis showed that IFN-alpha rapidly enhances mRNA expression of UbcH5, UbcH6 and UbcH8 in T cells. In addition, these genes were induced in macrophages in response to IFN-alpha or IFN-gamma stimulation or influenza A or Sendai virus infections. Similarly, IFNs enhanced UbcH8 mRNA expression in A549 lung epithelial cells, HepG2 hepatoma cells, and NK-92 cells. Cycloheximide, a protein synthesis inhibitor, did not block IFN-induced upregulation of UbcH8 mRNA expression, suggesting that UbcH8 is the primary target gene for IFN-alpha and IFN-gamma. Ubiquitin conjugation is a rate-limiting step in antigen presentation and therefore the upregulation of UbcHs by IFNs may contribute to the enhanced antigen presentation by macrophages. Our results show that proteome analysis of cells is a suitable method for identifying previously unrecognized cytokine-inducible genes.

Cells, Cultured↗

PubMed1998

Structural characterisation of N-linked and O-linked oligosaccharides derived from interferon-alpha2b and interferon-alpha14c produced by Sendai-virus-induced human peripheral blood leukocytes.

We have previously isolated and partially characterised the components of a highly purified interferon-alpha (IFN-alpha) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-alpha species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-alpha subtypes produced by Sendai-virus-induced human peripheral blood leukocytes, Biochem. J. 329, 295-302]. Here, we isolated the N-linked oligosaccharides of IFN-alpha14c and the O-linked chains of IFN-alpha2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-alpha14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10% carrying an additional alpha1,3-linked fucose unit at the antennae. The IFN-alpha2b was shown to carry about 50% core type-1 disialyltetrasaccharides, 30% core type-1 monosialyltrisaccharides and 20% core type-2 monosialylpentasaccharides.

Glycosylation↗

PubMed1998

Identification of nine interferon-alpha subtypes produced by Sendai virus-induced human peripheral blood leucocytes.

The human interferon-alpha (IFN-alpha) family is encoded by 13 different functional genes, and including all cloned sequence variants there are 28 potential IFN-alpha proteins. To find out which of the described sequences are expressed in normal human leucocytes, we have isolated and partly characterized the components of a highly purified IFN-alpha preparation produced by Sendai virus-induced human peripheral blood leucocytes. The identification protocol consisted of N-terminal sequencing and mass mapping of the proteins separated by reverse-phase HPLC and/or SDS/PAGE. The highly purified leucocyte IFN-alpha preparation was found to contain at least nine different IFN-alpha species: IFN-alpha1a, IFN-alpha2b, IFN-alpha4b, IFN-alpha7a, IFN-alpha8b, IFN-alpha10a, IFN-alpha14c, IFN-alpha17b and IFN-alpha21b. IFN-alpha1a was the major subtype, comprising approx. 30% of total leucocyte IFN-alpha. IFN-alpha14c, the only subtype containing potential N-glycosylation sites, was shown to be glycosylated at Asn-72. Molecular mass determination of the intact proteins by electrospray ionization MS showed that there are no other post-translational modifications in the IFN-alpha subtypes than the glycosylation of IFN-alpha2b and IFN-alpha14c. Only one sequence variant was found for each subtype, suggesting that the other described gene sequences represent allelic variants or mutations that are more rarely found in the general population.

Amino Acid Sequence↗