Biomedical subjects

Martin Kussmann

Publications and source records attributed to Martin Kussmann.

11 recordsLinked to original sources

PubMed2006

Automated target preparation for microarray-based gene expression analysis.

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.

Animals↗

PubMed2006

OMICS-driven biomarker discovery in nutrition and health.

While traditional nutrition research has dealt with providing nutrients to nourish populations, it nowadays focuses on improving health of individuals through diet. Modern nutritional research is aiming at health promotion and disease prevention and on performance improvement. As a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. Nutrigenetics asks the question how individual genetic disposition, manifesting as single nucleotide polymorphisms, copy-number polymorphisms and epigenetic phenomena, affects susceptibility to diet. Nutrigenomics addresses the inverse relationship, that is how diet influences gene transcription, protein expression and metabolism. A major methodological challenge and first pre-requisite of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (protein expression analysis) and metabonomics (metabolite profiling) to define a "healthy" phenotype. The long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. Transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. Metabonomics is a diagnostic tool for metabolic classification of individuals. The great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. This feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. Apart from addressing the most complex "-ome", proteomics represents the only platform that delivers not only markers for disposition and efficacy but also targets of intervention. The Omics disciplines applied in the context of nutrition and health have the potential to deliver biomarkers for health and comfort, reveal early indicators for disease disposition, assist in differentiating dietary responders from non-responders, and, last but not least, discover bioactive, beneficial food components. This paper reviews the state-of-the-art of the three Omics platforms, discusses their implication in nutrigenomics and elaborates on applications in nutrition and health such as digestive health, allergy, diabetes and obesity, nutritional intervention and nutrient bioavailability. Proteomic developments, applications and potential in the field of nutrition have been specifically addressed in another review issued by our group.

Animals↗

PubMed2006

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification.

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.

Animals↗

PubMed2006

Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.

Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

Caseins↗

PubMed2006

Proteomic methods in nutrition.

PURPOSE OF REVIEW: Proteomics, the comprehensive analysis of a protein complement in a cell, tissue or biological fluid at a given time, is a key player in the family of -omic disciplines, which encompass genomics (gene analysis), transcriptomics (gene expression analysis) and metabolomics (metabolite profiling). This review summarizes the state of the art of proteomics technology and puts it into perspective for food-related research. Learning from proteomic experiences in the pharmaceutical context, this article may help to translate proteomics into nutrition and health. RECENT FINDINGS: Mass spectrometric technology has progressed enormously with regard to mass accuracy, resolution and peptide sequencing power. Likewise, upstream separation, depletion and enrichment techniques now allow us to deal with the large complexity and wide dynamic range of proteomic samples more efficiently. Consequently, proteomic studies now provide a broader, but still far from complete, coverage of a given proteome. SUMMARY: Proteomics adapted and applied to the context of nutrition and health has the potential to deliver biomarkers for health and comfort, reveal early indicators of disease disposition, assist in differentiating dietary responders from non-responders, and, last but not least, discover bioactive, beneficial food components.

Biomarkers↗

PubMed2005

Rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by multidimensional protein identification technology.

Direct injection of complex protein mixtures, e.g. those derived from crude biological fluids, is often incompatible with conventional LC supports, because of column clogging and rapid deterioration of chromatographic performance. In this paper, we report the use of restricted access media to rapidly enrich and fractionate human breast milk. This resin, combining size exclusion and anion exchange functionalities, yielded a fraction enriched in soluble CD14 and showing specific sCD14-dependant activity. This fraction was split into five aliquots, which were individually characterized using multidimensional protein identification technology. Reproducibility of the results was addressed by analysing and comparing five datasets using different protein identification tools available within the Sequest software. Furthermore, a comparison of three major releases of the Ensembl human protein database was performed to examine the effect of database updates on our results. We report here the benefit of repeated analysis of aliquots of the same fraction: first to increase the confidence in peptide identification by repeated confirmation in several aliquots; and second to assess experimental reproducibility. We demonstrate furthermore the effect of database modifications on the results and the importance of constantly re-analysing data with new releases to keep them consistent and up to date with the latest protein identities and predictions available.

Amino Acid Sequence↗

PubMed2005

Proteomics of the rat gut: analysis of the myenteric plexus-longitudinal muscle preparation.

The enteric nervous system (ENS)--present all along the gastrointestinal tract - is the largest and most complicated division of the peripheral nervous system that can function independently of the brain. The peripheral nerve cells are organized in two separate but interconnected meshworks, called the myenteric and submucous plexus. The nervous control of intestinal motility is primarily governed by the myenteric plexus (MP), which lies in-between the longitudinal- (LM) and circular-muscle layers and regulates their functions. To determine whether the proteomic technology is adapted to the analysis of specific gut tissues, we dissected the MP-LM layers from the jejunum, ileum, and colon of Long Evans rats, homogenized them, and separated the proteins using two-dimensional gel electrophoresis. A subset of all the visualized protein spots, covering the entire range of molecular weights and isoelectric points, was then selected and further analyzed by matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry. We identified around 80 proteins in each gut segment, and among those, five were segment-specific. Most of the proteins identified were derived from muscle cells, but we also detected some neuron-specific proteins. This study represents, to our knowledge, the first extensive protein catalog of a neuromuscular layer of the rat intestine and it may constitute the basis to understand pathophysiological mechanisms related to the ENS.

Animals↗

PubMed2005

Proteomics in nutrition and health.

Proteomics, the comprehensive analysis of a protein complement in a cell, tissue or biological fluid at a given time, has been enabled by quantum leaps in mass spectrometric technology, which allowed identification of large, involatile biomolecules. Over the last two decades, this discipline evolved from the sole delivery of protein identities to a platform, which reveals clues to function through e.g. characterisation of protein modifications and interactions as well as through quantitative proteomics, i.e. the global comparison of protein amounts between two defined biological states. Proteomics is an integral part and key player in the family of -omic disciplines as there are genomics (gene analysis), transcriptomics (gene expression analysis) and metabolomics (metabolite profiling). Considering the complexity, dynamics and protein concentration range of any given proteome, proteomics is the most challenging -omic discipline and requires the most sophisticated analysis pipeline. Proteomics represents an established technology in the pharmaceutical industry mainly for biomarker and drug target discovery. The potential of proteomics for research in the food industry is increasingly being recognised and the employment of proteomic approaches to nutrition and health issues is now emerging. This review summarizes (i) major technological achievements in mass spectrometry and proteomics, (ii) deliverables of proteomics in the context of nutrition and health, and (iii) applications of proteomics, and -- if appropriate -- transcriptomics to the research fields of digestive health, obesity and diabetes, immunity and allergy, probiotics, milk, and food preference.

Complex Mixtures↗

PubMed2004

Identification of protein-protein interfaces implicated in CD80-CD28 costimulatory signaling.

The B7 ligands CD80 and CD86 on APCs deliver either costimulatory or inhibitory signals to the T cell when interacting with their counter-receptors CD28 and CD152 (CTLA-4) on the T cell surface. Although crucial for lymphocyte regulation, the structural basis of these interactions is still not completely understood. Using multivalent presentation and conditions mimicking clustering, believed to be essential for signaling through these receptors, and by applying a combined differential mass spectrometry and structural mapping approach to these conditions, we were able to identify a putative contact area involving hydrophilic regions on both CD28 and CD80 as well as a putative CD28 oligomerization interface induced by B7 ligation. Analysis of the CD80-CD28 interaction site reveals a well-defined interface structurally distinct from that of CD80 and CD152 and thus provides valuable information for therapeutic intervention targeted at this pathway, suggesting a general approach for other receptors.

Animals↗

PubMed2004

Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

Amino Acid Sequence↗

PubMed2004

Rapid identification of stress-related fingerprint from whole bacterial cells of Bifidobacterium lactis using matrix assisted laser desorption/ionization mass spectrometry.

Whole cells of Bifidobacterium lactis were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Characteristic and reproducible mass spectra were obtained in the mass range from 6 to 19 kDa. After several days of bacterial cell storage at 4 degrees C (D0, D2, and D6), only minor signal differences were observed. Under identical and reproducible conditions, fourteen relevant diagnostic ions were identified. Moreover, control- and stress-related fingerprints were rapidly obtained using MALDI-TOFMS by comparison of protein patterns obtained from non-stressed (control) versus stressed cells (addition of bile salts during growth). After quantitative validation of the MALDI-MS data by a statistical approach, two and eight signals were assigned as control- and stress-specific ions, respectively. This work provides the evidence that MALDI-TOFMS can be used for the identification of stress-related fingerprint of B. lactis bacterial cells and could have a high potential for the assessment of the physiological status of the cells.

Bifidobacterium↗