Biomedical subjects

Hanno Steen

Publications and source records attributed to Hanno Steen.

22 records · Page 2Linked to original sources

PubMed2002

A new derivatization strategy for the analysis of phosphopeptides by precursor ion scanning in positive ion mode.

Although numerous strategies have been devised to analyze protein phosphorylation, an abundant intracellular protein modification, there is still a need for different methods for the analysis of this modification. A method to both detect and localize the phosphorylation within a protein/peptide is especially required. In this paper, a new strategy is described, which makes use of beta-elimination/Michael addition reactions to introduce a functional group at the original site of phosphorylation, which gives rise to a dimethylamine-containing sulfenic acid derivative with a unique m/z value. This enables the detection of the phosphorylated species within peptide mixtures by sensitive and specific precursor ion scanning in positive ion mode. Working under acidic conditions in positive ion mode has the added advantage that subsequent normal peptide sequencing for the exact localization can be performed. No other peptide derived fragment ion is observed at the m/z value of the sulfenic acid derivative formed, thus specific precursor ion experiments can also be carried out on instruments with low fragment ion resolution and lends itself to LC-MS/MS approaches when skimmer fragmentation routines or triple quadrupole mass spectrometers are used.

Calmodulin↗

PubMed2002

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

3T3 Cells↗

PubMed2001

Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway.

Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.

ErbB Receptors↗

PubMedDate not supplied

Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry.

Photochemical cross-linking is a commonly used method for studying the molecular details of protein-nucleic acid interactions. Photochemical cross-linking aids in defining nucleic acid binding sites of proteins via subsequent identification of cross-linked protein domains and amino acid residues. Mass spectrometry (MS) has emerged as a sensitive and efficient analytical technique for determination of such cross-linking sites in proteins. The present review of the field describes a number of MS-based approaches for the characterization of cross-linked protein-nucleic acid complexes and for sequencing of peptide-nucleic acid heteroconjugates. The combination of photochemical cross-linking and MS provides a fast screening method to gain insights into the overall structure and formation of protein-oligonucleotide complexes. Because the analytical methods are continuously refined and protein structural data are rapidly accumulating in databases, we envision that many protein-nucleic acid assemblies will be initially characterized by combinations of cross-linking methods, MS, and computational molecular modeling.

Amino Acid Sequence↗