Current Pharmaceutical Design - Volume 11, Issue 20, 2005

Recent developments in the field of mass spectrometry, especially in the instrumentation (MALDI and ESI ion sources) and electronic improvements in the past decade have placed this technique at the forefront of many biological applications. Recent studies performed these last years on the proteome have demonstrated the usefulness of this technology. The combination of mass spectrometry with classical biochemical methods has lead to the determination and identification of different proteins using proteomics studies in close relation with genome databases. Although such methods generally lead to the identification of novel proteins, they do not allow determining the cellular localization or regulation of peptide/proteins expression in tissues, cellular groups or single cells. New emerging technologies enable the development of alternative methodologies to address such questions. This is the aim of this issue of Current Pharmaceutical Design devoted of 6 reviews on mass spectrometry techniques from biomolecules (peptide/protein, DNA, glycosylation) analysis to Imaging assisted laser desorption/ionization (MALDI), laser desorption mass spectrometry. The first article from G. Bolbach [1] (France) aims to give an overview of MALDI mass spectrometry and its applications for the analysis of non covalent complexes. It will focus on the condition for generating intact complexes, keeping in mind the present knowledge of fundamental processes. It will also describe its combination with specific chemical, biochemical and biological methodologies and strategies to address the fascinating role of such association in living systems The second review From C. Afonso and Colleagues [2] (France) is focused on the various surface modifications used in combination with laser desorption mass spectrometry and their application for the analysis of peptides and proteins. In the first hand, some modified surfaces are designed to enhance the laser desorption/ionization process, this includes the use of carbon, porous silicon surfaces and also immobilized matrix. In another hand chemical and biochemical modified surfaces developed to isolate species with more or less specific interactions can be used for on-slide sample clean-up before MALDI-MS analysis. In addition the different experimental devices as mass spectrometers and microfluidic devices used for such a purpose will be presented. The third review from W. Pusch and M. Kostrzewa [3] (Germany) gave an overview of the application of mass spectrometry in the fields of screening and diagnostic research. It point on the used of mass spectrometry methods to enable a very early diagnosis of diseases with minimally invasive methods of investigation. This type of high end screening application will have the potential to revolutionize the early diagnosis of many diseases. The fourth paper from J.L. Frahm and D.C. Muddiman [4] (USA) is a complete review on Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry analysis of DNA, RNA and peptide nucleic acid. The fifth paper from W. Morelle and J.C. Michalsky [5] (France) is devoted to the research field of glycobiology: the glycomics. The last one from S. Vandevoorde and D.M. Lambert [6] (Belgium) is a review disconnected to the focus of the mass spectrometry issue but focus on the Enzyme inhibition. References [1] Bolbach G. Matrix-Assisted Laser Desorption/Ionisation Analysis of Non-Covalent Complexes: Fundamentals and Applications. Curr Pharm Design 2005; 11(20): 2535-2557. [2] Afonso C, Budimir N, Fournier F, Tabet J-C. Activated Surfaces for Laser Desorption Mass Spectrometry: Application for Peptide and Protein Analysis. Curr Pharm Design 2005; 11(20): 2559-2576. [3] Pusch W, Kostrzewa M. Application of MALDI-TOF Mass Spectrometry in Screening and Diagnostic Research. Curr Pharm Design 2005; 11(20): 2577-2591. [4] Frahm JL. Muddiman DC. Nucleic Acid Analysis by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry at the Beginning of the Twenty-First Century. Curr Pharm Design 2005; 11(20): 2593-2613. [5] W. Morelle and Michalski J-C. Glycomics and Mass Spectrometry. Curr Pharm Design 2005; 11(20): 2615-2645. [6] Vandevoorde S, Lambert DM. Focus on the Three Key Enzymes Hydrolysing Endocannabinoids as New Drug Targets. Curr Pharm Design 2005; 11(20): 2647-2668.

Mass spectrometry is now a well established and powerful method for analysing the structure of biomolecules and more recently their non-covalent interactions. It is based on two soft ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), which allow to produce biomolecule ions in gas phase with a very high efficiency for a subsequent analysis in the mass analyser. The high sensitivity and the high precision of modern mass spectrometry were first applied to elucidate the primary structure of proteins in the so-called proteomic area. It is widely used in biological and medical sciences. The new challenge for the mass spectrometrist is to decipher the quaternary structure of biomolecules by preserving into the gas phase the weak interactions that exist between molecules in solution. In this review, we will focus on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) for studying the non-covalent associations. Keeping in mind that the weak interactions between partners must be preserved from the solution into the gas phase, we will describe the critical parameters involved in both the desorption/ionization processes and the target preparation including the incorporation of the non-covalent complex into the matrix crystals. Various examples will be presented showing that weak interactions can survive the entire MALDI process allowing the direct detection of intact complexes. However, all these weak interactions cannot be always preserved from the cell to the mass spectrometer. Specific methodologies have been developed to get insights of these interactions. Most of them will be presented in this review.

Thanks to the development of matrix assisted laser desorption/ionisation (MALDI), laser desorption based mass spectrometry became an essential method for the analysis of biomolecules. This review will discuss the various surface modifications used in combination with laser desorption mass spectrometry and their application for the analysis of peptides and proteins. In the first hand, some modified surfaces are designed to enhance the laser desorption/ionisation process; this includes the use of carbon, porous silicon surfaces and also immobilised matrix. In an other hand chemical and biochemical modified surfaces developed to isolate species with more or less specific interactions can be used for onslide sample clean-up before MALDI-MS analysis. In addition, different experimental devices as mass spectrometers and microfluidic devices used for such a purpose will be presented.

During the last years, mass spectrometry has revolutionised protein biochemistry and has advanced to a superior tool for the identification and detailed analysis of peptides and proteins. The high throughput allowed by some mass spectrometry platforms has enabled the important step from analysis of individual proteins to proteomics. Recently, an additional field of mass spectrometry applications has emerged - namely screening and diagnostic research. In contrast to protein identification, screening applications have to detect analyte molecules of defined molecular weights which can be calculated beforehand, for example by means of chemical structures. Here, the accuracy and sensitivity of mass spectrometry has to be combined with the requirements of high-throughput analyses, in particular speed and automation. These criteria are especially fulfilled by state of the art matrix-assisted laser desorption/ionisation time-offlight mass spectrometry (MALDI-TOF MS) instruments. The first high throughput screening (HTS) application proved to be genotyping of single nucleotide polymorphisms. The same principle was later applied for several quality control issues, for example for oligonucleotides, peptide or compound libraries. This development has culminated in the screening and profiling of complex biomarker patterns in clinical proteomics to detect a molecular fingerprint for specific diseases in biological samples. Thus, mass spectrometry based methods are expected to enable a very early diagnosis of diseases with minimally invasive methods of investigation. This type of high end screening application has the potential to revolutionise the early diagnosis of many diseases. Here, we give an overview of the application of mass spectrometry in the fields of screening and diagnostic research.

Mass spectrometers measure an intrinsic property (i.e., mass) of a molecule, which makes it an ideal platform for nucleic acid analysis. Importantly, the unparalleled capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry further extend its usefulness for nucleic acid analysis. The beginning of the twenty-first century has been marked with notable advances in the field of FT-ICR mass spectrometry analysis of nucleic acids. Some of these accomplishments include fundamental studies of nucleic acid properties, improvements in sample clean up and preparation, better methods to obtain higher mass measurement accuracy, analysis of noncovalent complexes, tandem mass spectrometry, and characterization of peptide nucleic acids. This diverse range of studies will be presented herein.

Proteomics is closely associated with the modifications of the gene product such as the post-translational events that yield functionally active gene products. Among these, glycosylation represents a critically important posttranslational modification and is a target for proteomic research. Glycan moieties are involved in a wide variety of intracellular, cell-cell and cell-matrix recognition events. This is why understanding how glycosylation affects the activities and functions of proteins in health and disease represents a major challenge. The study of the glycome - the whole set of glycans produced in a single organism - is therefore essential to determine the functions of all genes. Mass spectrometry, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of oligosaccharides. This review provides a summary of the current knowledge for the mass spectrometric analysis of glycoproteins and their glycan structures.

The family of endocannabinoids (i.e., the endogenous agonists of cannabinoid receptors) contains several polyunsaturated fatty acid amides such as anandamide (AEA) and oleamide but also esters such as 2-arachidonoylglycerol (2-AG). These compounds are the subject of growing interest in pharmacology for their multiple therapeutic potentials. Unfortunately, they are rapidly inactivated by enzymatic hydrolysis, which prevents their effective medical use. Inhibitors of endocannabinoid degradation seem to be necessary tools for the development of endocannabinoid therapeutics. But hitting this target is inconceivable without good knowledge of the enzymes. Fatty acid amide hydrolase (FAAH) is the oldest and the best characterised enzyme involved in the degradation of endocannabinoids. Cloning, distribution in the body and crystal structure of FAAH have been described. A large number of FAAH inhibitors have also been synthesised and tested. For a long time, FAAH was considered as the only key enzyme hydrolysing endocannabinoids. But recent findings indicate that at least two other enzymes have critical role in the endocannabinoids degradation. Monoglyceride lipase participates in 2-AG degradation and some data indicate that it is the primary mechanism for 2-AG inactivation in intact neurons. N-palmitoylethanolamine-selective acid amidase (NPAA) is a second fatty acid amide hydrolase more active with N-palmitoylethanolamine, an anti-inflammatory substance. The purpose of this review is to collect and compare the catalytic properties of these 3 key enzymes hydrolysing endocannabinoids.