Biomedical subjects

Christian W Huck

Publications and source records attributed to Christian W Huck.

21 records · Page 2Linked to original sources

PubMedDate not supplied

Development and evaluation of an in vitro model for the analysis of cigarette smoke effects on cultured cells and tissues.

INTRODUCTION: Smokers have an increased risk for a variety of diseases. Among the most prominent is atherosclerosis, the leading cause of death in the Western world. Although this conjunction is accepted knowledge, the basic biological mechanisms and the identities of the active tobacco smoke constituents surprisingly are still unknown. One reason for this is the lack of accurate in vitro models. METHODS: Cell culture experiments, including cell morphology and cell death analyses, high-performance liquid chromatography, and liquid chromatography coupled to mass spectrometry via an electrospray ionization interface allowing collision-induced dissociation analyses, were applied. RESULTS AND DISCUSSION: In this study, we present and validate an in vitro model that has proven to be useful for standardized studies of cellular and histological effects of cigarette smoke. The system consists of a cigarette smoke sampling device in which water-soluble cigarette smoke constituents pass over from the gas phase into the aqueous phase resulting in nicotine concentrations identical to the in vivo concentrations, suggesting in vivo similar conditions for gas-to-liquid compound exchange.

Cell Survival↗

PubMedDate not supplied

Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics.

The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).

Amino Acid Sequence↗

PubMedDate not supplied

Derivatized cellulose combined with MALDI-TOF MS: a new tool for serum protein profiling.

Providing a rapid and sensitive protein profiling method for biomarker discovery from a variety of biological samples is crucial for the introduction of new markers that improve cancer patient diagnosis at early tumor stages, thus increasing the chances of curative treatment. We report here the development and application of derivatized cellulose particles for selective serum protein profiling. For immobilized metal ion affinity chromatography (IMAC), cellulose was derivatized with glycidyl methacrylate (GMA) and subsequently with iminodiacetic acid (IDA). To investigate the application of this material for generating protein profiles of human serum samples, the serum samples were agitated with the derivatized cellulose particles to a suspension and incubated for 2 h at 30 degrees C. After washing, 1 microL of the IDA-Cu(2+)-cellulose suspension was applied directly onto a MALDI-target, mixed with sinapinic acid (SA) and analyzed with MALDI-TOF MS. Consistent serum specific data were obtained from aliquoted samples analyzed several times, indicating the reliability of the method. However, the serum fingerprints obtained proved to be specific for any given serum. The technique presented allows a high enrichment of sample on the developed target leading to a high sensitivity and reproducibility without depletion of albumin and immunoglobulin, and sample elution prior to MS-analysis. The study demonstrates for the first time that derivatized cellulose particles combined with MALDI-TOF MS represent a simple, economical, and rapid approach to generate serum protein profiles for biomarker identification.

Biomarkers↗