Mappa Humanus | Mappa Humanus - A Visionary Venture |
|
The Mappa Humanus is a strategic initiative to map and spectrally characterize human metabolic components using various mass spectrometry and hyphenated techniques. This visionary project employs an innovative approach to addressing fundamental issues associated with the compositional and comparative analysis of the small, but structurally extremely diverse biomolecules in complex biological matrices. The resulting map of endogenous metabolites and their spectral fingerprints will lay the foundations for future metabolomic (metabonomic), biomedical and pharmaceutical research aimed at comprehensive, earlier and more precise diagnosis, prevention strategy and effective treatment to relieve the symptoms of a wide variety of human diseases. To meet these challenges a new integrated approach is being developed, which will address acquisition, processing and, above all, the interpretation of the vast amount of mass spectrometric data needed to accomplish the ultimate goal of mapping the human metabolome.
The Concept The overall concept is based on the systematical analysis of all detectable metabolic compounds in human body. This concept poses an enormous analytical challenge reaching beyond existing strategies and will require the development of novel methodologies aimed at the detection, identification and characterization of numerous components in complex mixtures. A number of advanced technologies will be applied to acquire reliable and comprehensive analytical data from biological fluids, tissues and organs, to process the vast amount of resulting heterogeneous data, and build a computer-based platform for data interpretation. To achieve this objective, we will apply high resolution tandem mass spectrometry instrumentation and our leading, proprietary state-of-the-art software technology which utilizes numerous advanced algorithms together with libraries of empirical data. It will be necessary to combine these technologies in order to identify and fingerprint human metabolic components. Metabolomics (Metabonomics) Today, a new paradigm is taking place - a move from understanding the mechanisms of life based on genomics, transcriptomics and proteomics to mapping endogenous metabolites and comprehending their functions, levels and role in system biology. The emerging science, referred to as “metabolomics” (metabonomics) is the study of small molecules that can be expressed by a living organism. Representative groups of these small molecules include vitamins, lipids, sugars, organic acids and steroids. Metabolomics directly reflects the current physiological status of an organism in contrast to genetics, which is hardwired and plugged into the organism from birth. The applications of metabolomics includes disease biomarkers discovery, target identification and validation, monitoring and evaluation of therapeutic intervention, clinical diagnostic and a “personalized medicine” vision. Metabolomics has enormous clinical potential as an organism’s metabolic profile closely reflects its current fitness and not merely potential changes in biological activities or a predisposition to phenotypic abnormalities as is the case for genomics. The pharmaceutical industry views metabolomics as a promising technology for “rational” drug design. Disease targets are being identified using differential metabolic expressions between normal - disease profiles (metabotyping) and subsequent target validation also requires the investigation of metabolic regulation and fluxes. The metabolomic approach provides a powerful new tool for gaining insights into functional biology which correlate interdependencies between gene expression, protein activities, metabolite levels and morphology.
Why Mass Spectrometry? The challenge set by metabolomic research is the analysis of numerous small molecules in background rich biological matrices over a dynamic range of ten orders of magnitude. A highly sensitive, selective and specific analytical technique is required to study the complex mixture of metabolites within a cell or a biofluid. Mass spectrometry has become the method of choice for emerging metabolomic applications for the structural, quantitative and differential analysis of low and high molecular species. Unparalleled levels of sensitivity, combined with a wealth of structural information, makes mass spectrometry an indispensable bioanalytical research tool. The superior sensitivity of mass spectrometry means it is likely to be increasingly favored over NMR in the light of the trend towards biofluid analysis rather than intact tissues due to their non- or minimal invasive sample collection. Within this context, the nondestructive nature of NMR becomes less important. The relatively low cost of the instrumentation represents a further advantage of mass spectrometry and it is unlikely that the more expensive NMR will become a routine analytical technique in the foreseeable future even in an economically interesting life science sector. Although NMR is a superior qualitative and quantitative technique, it is of no use for one area of particular interest – profiling of “cell leaking”, extremely low abundant metabolites, due to its low sensitivity and resulting inability to detect those components. Structural Work Metabolomics (metabonomics) includes a variety of applications, many of which ultimately end up with structural assignments of individual metabolic components. Structural work in mass spectrometry relies on two principal methods: compound identification using spectra search in reference libraries and spectra interpretation. Spectra search is a popular method in certain application areas (environmental, forensic, etc.) but there are several potential complications regarding bioanalytics. The relevant publicly available databases are limited to GC/MS and many biomolecules are not amenable to this technique without prior derivatization due to their insufficient thermal stability and low volatility. The emerging LC/MS libraries continue to be incomprehensive, and are mostly oriented towards existing and potential pharmaceuticals, and often lack specificity because of their “soft” nature. Larger and more homogenous LC/MS libraries need to be built along with supplementary experimental parameters needed for advanced search algorithms. Using a combination of hyphenated techniques and mass spectrometry, data can be generated in terabyte volumes at a high-throughput speed. However, contemporary interpretation techniques require human interaction, supplementary structural information and/or reference spectra of structurally related compounds to unambiguously determine the composition and structural arrangement of an unknown metabolite. Spectra interpretation of unknowns frequently presents a serious bottleneck because of the vast structural diversity of small molecules and the complexity of mass spectral data. Even though mass spectrometry provides information rich spectra, the structural arrangement is ciphered through the set of fragment masses and in some rare cases a confident structure determination without additional pieces of structural information is currently only possible using different structure elucidation techniques, mainly NMR. We believe that this obstacle can be overcome using a novel systematic approach which will decipher the structural information of biologically related molecules from mass spectra based on our proprietary technology. The Objective Comprehensive identification of endogenous metabolites and the creation of their spectral signatures promises to have a significant impact on metabolomic (metabonomic) research. Instantaneous identification and selective quantitation of metabolic components will facilitate the routine application of mass spectrometry in many fields of metabolomics, particularly human healthcare research. In all the stages of biomarker discovery, it is an immense advantage to know the identities of differentially expressed metabolic components compared to viewing them solely as statistical objects. Prior structural knowledge allows the exclusion of random, uncharacteristic or confounding metabolites in the classification process based on accumulated knowledge from biochemical pathways, or from previously conducted experiments. Instantaneous identification of molecular species that differentiate the pathological from the normal provides the required speed and confidence particularly when more common multicomponent biomarkers are identified rather than any one, single dominant biomarker. Not only the effectiveness and resulting cost saving, but sometimes even the ability to carry out a structural determination of low abundant metabolic components without a reference library can represent a serious issue. The pharmaceutical industry considers metabolomics (metabonomics) as a promising area with regard to target identification and validation as well as drug safety and efficiency. Structural elucidation of endogenous and xenobiotic metabolites remains the major bottleneck for high-throughput strategies, creating increasing demand for comprehensive reference libraries. Spectral and structural libraries of the human metabolome can cover an important portion of compounds encountered in the course of drug discovery and drug development work. By definition, metabolomics studies changes in the concentrations and fluxes of endogenous metabolites in response to a stressor - intervention or pathophysiological stimuli. Quantitation poses a real analytical challenge because the human metabolome has an extraordinary dynamic range of more than 9 orders of magnitude separate glucose and the rarest metabolites now detected. Despite the known complication of ion suppression, mass spectrometry has the potential to become a mature, robust and reliable quantitative technique in routine metabolomics utilizing information on stage dependent ion profiles of individual metabolites obtained from spectral libraries. As a result, spectral libraries can serve as an indispensable source of information in planning analytical strategies for the use of standard quantitation methods such as single and multiple reaction monitoring, single ion monitoring or the simple comparison of base peak intensities. Compound specific peak(s) picking, simplifying integration, minimizing the potential for collision-cell cross talk, preventing interferences where libraries can contribute to establishing a quantitative method and making this process more automatic. The compositional aspect of the characterized human metabolome may facilitate a better understanding of biology at the system level. Qualitative knowledge of human metabolites can serve as a basis for the topological mapping of missing metabolic pathways raring to go for screening against unannotated proteins to which no apparent function could be assigned. The upstream process is likely to continue toward translational and genomic studies by taking a global approach to gaining a deeper functional and mechanistic insight into cellular processes. Finally, the technology developed by the Mappa Humanus project could pave the way for faster, cheaper and more effective identification of unknown metabolites in natural and genetically modified organisms. While many different species may express and utilize essentially identical metabolic pathways, the spectral fingerprint approach promises to yield significantly more straightforward metabolic reconstruction than current genome deducing methods.
|
