Biomedical subjects

Christoph Roesli

Publications and source records attributed to Christoph Roesli.

4 recordsLinked to original sources

PubMed2006

A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer.

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

Antigens, Neoplasm↗

PubMed2006

Two-dimensional mass spectrometric mapping.

The identification and relative quantification of proteins in closely related biological samples is the backbone for many investigations in systems biology and for the discovery of biomarkers. While two-dimensional gel-based methodologies are still widely used for comparative proteomic studies, the recent advent of gel-free methodologies may allow the analysis of a larger number of samples in an automated fashion. Most of the technologies presented in this review require a chemical modification of proteins before analysis, and rely on the relative intensities of mass spectrometry signals for protein quantification. In particular, two-dimensional mass spectrometric mapping methodologies provide a visual representation of mass spectrometric data, thus facilitating the identification of differences in relative protein abundance.

Humans↗

PubMed2005

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping.

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.

Biotinylation↗

PubMed2005

A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins.

2-D peptide mapping is a novel technique for the relative quantification of membrane proteins (Scheurer S. et al., Proteomics 2005, in press). Using closely related metastatic and nonmetastatic teratocarcinoma cell lines as a model system, we have performed a comparative analysis of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques, with the aim to increase the number of proteins identified by 2-D peptide mapping. Our experience indicates that the LC-MALDI TOF/TOF technique is superior to LC-ESI MS/MS in terms of the number of proteins identified and confidence in protein identification. Furthermore, the best results were obtained by tryptic digestion of proteins eluted from a streptavidin column using a cleavable biotin derivative.

Animals↗