Agnieszka Kraj

Morphinome – proteome of the nervous system after morphine treatment

Summary.Proteome is a natural consequence of the post-genome era when the HUGO project (Human Genome Organization) has almost been completed. Here, a specifically aimed proteome in drug dependence – morphinome, is described, including tasks, strategies and pitfalls of the methodology.

Desorption/ionization mass spectrometry on array of silicon microtips

We have shown that a sample of bio-species deposited onto the spot of the array of protruded metal gated silicon tips (DIOST) may be effectively ionized and evaporated by UV laser pulsed light illumination. This phenomenon has been never observed on flat surface of silicon dioxide or TiW-Au thin film layers the materials which were used in construction of the tips array. Nature of this process is not known; the most intriguing fact is the protonization as well as negative electron ionization of bio-samples. The new DIOST platform was used for TOF MS tests, and clearly readable, low-noised mass spectrograms of LGG and dopamine were observed.

Fingerprinting of 3, 4-Methylenedioxymethamphetamine Markers by Desorption/Ionization on Porous Silicon

Desorption/ionization on porous silicon (DIOS) is a method which extends the application range of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This technique eliminates matrix background in the low mass range; DIOS is especially advantageous in research on small organic molecules and their metabolites in biological samples. DIOS mass spectrometry was applied for 3, 4-methylenedioxymethamphetamine, (MDMA, Ecstasy) impurities identification. Trace component profiling enables the identification of by-product characteristics for the synthesis route of MDMA. Ecstasy, a synthetic psychoactive drug, is highly popular among young people, and often used as a recreational drug, most commonly during disco parties. MDMA enhances feeling of euphoria by increasing the level of neurotransmitters such as serotonin, dopamine and norepinephrine and causes acute behavioral and psychological effects. MDMA is almost exclusively produced illegally, primarily in Western Europe. The new method for MDMA impurities profiling has been developed to trace the origin of MDMA pills. For comparison and classification of the impurity profiles, principal components analysis was used.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid

Dynorphin‐converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin‐related peptides bind to δ‐opioid instead of κ‐opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin‐converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The KM value of the convertase was found to be 9 µm, and the Ki value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

Desorption/ionization mass spectrometry on array of silicon micro tips

We have shown that a sample of bio-species deposited onto the spot of the array of protruded metal gated silicon tips (DIOST) may be effectively ionized and evaporated by UV laser pulsed light illumination. This phenomenon has been never observed on flat surface of silicon dioxide or TiW-Au thin film layers the materials which were used in construction of the tips array. Nature of this process is not known; the most intriguing fact is the protonization as well as negative electron ionization of bio-samples. The new DIOST platform was used for TOF MS tests, and clearly readable, low-noised mass spectrograms of LGG and dopamine were observed.

Chapter 24 Computer resources

Publisher Summary This chapter discusses resources that can be mustered from a computer and lists Websites that are or might be useful in a laboratory, such as software for chromatography, electrophoresis, and mass spectrometry, literature databases, tutorials, instrumentation, vendors, journals, and software for proteomics/genomics. The chapter presents a list of companies/manufacturers of scientific and analytical instrumentation, chemicals, and software, as well as representatives, who may be contacted for these Websites. The chapter discusses various aspects of mass spectrometry, coupling with commonly used chromatographic/electrophoretic techniques, tutorials, procedures, methods for spectral interpretation, troubleshooting, newsgroups, and other important links. Information related to manufacturers, resources, databases, tutorials, tools for identification, available software, and news for proteomics, genomics, and informatics are also presented in the chapter.

Chapter 5 Size-exclusion chromatography

Publisher Summary Size-exclusion chromatography (SEC), also called “gel-permeation chromatography,” “molecular-sieve chromatography,” or “gel filtration,” separates molecules according to their sizes. Smaller molecules are retarded on a column, whereas larger ones are eluted more rapidly. As the retention time can be directly correlated with the sizes of molecules, this method is particularly useful for the determination of molecular weight. SEC is generally applicable to the separation of molecules in the range of 0.5–1000 kDa, but larger proteins or other giant molecules can also be separated. SEC can also physically separate folded macromolecules from the unfolded ones, particularly in the case of slow equilibria. The major applications of SEC include the determination of molecular weight and molecular-weight distribution of natural and synthetic polymers. Separation strongly depends on many factors such as column packing, column dimensions, flow rate, sample volume, and mobile-phase composition. Capillary SEC provides a separation strategy for microscale purification. Capillary SEC columns are best suited for direct coupling to electrospray ionization–mass spectrometry (ESI–MS) because they have comparable flow rates that can be delivered to the ESI source without splitting.