Covalent adducts of melphalan with free amino acids and a model peptide studied by liquid chromatography/tandem mass spectrometry.

RATIONALE: Melphalan is a frequently used chemotherapeutical agent for the treatment of myeloma, breast cancer, ovarian cancer and sarcoma of soft tissue. A good knowledge of the reactivity of the drug toward the different amino acids, e.g. covalent adduct formation, is crucial for the understanding of its activity and side effects during cancer treatment. METHODS: The reactivity of melphalan and sites of adduct formation were studied by in vitro incubation of melphalan with free amino acids and glutathione as a model peptide. The formed covalent adducts were investigated using ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) using a triple-quadrupole instrument. Accurate mass measurements for the confirmation of characteristic product ions were performed on a quadrupole time-of-flight (QTOF) mass spectrometer. RESULTS: The incubation of melphalan with different classes of amino acids resulted in the formation of adducts on the amino and carboxyl termini, as well as adduct formation in the reactive side chains of Cys, Met, Tyr, His, Lys, Asp and Glu. All these melphalan adducts could be identified by their characteristic collision-induced dissociation (CID) product ion patterns. CONCLUSIONS: The present study demonstrates the reactivity of melphalan towards the functional groups of amino acids. The different alkylation site products show distinctive fragmentation patterns, which enable a fast identification of the different melphalan adducts. This study is a first important step towards a better understanding of the adduct formation in more complex molecules, e.g. peptides and proteins.

Aptamer-ligand recognition studied by native ion mobility-mass spectrometry.

The range of applications for aptamers, small oligonucleotide-based receptors binding to their targets with high specificity and affinity, has been steadily expanding. Our understanding of the mechanisms governing aptamer-ligand recognition and binding is however lagging, stymieing the progress in the rational design of new aptamers and optimization of the known ones. Here we demonstrate the capabilities and limitations of native ion mobility-mass spectrometry for the analysis of their higher-order structure and non-covalent interactions. A set of related cocaine-binding aptamers, displaying a range of folding properties and ligand binding affinities, was used as a case study in both positive and negative electrospray ionization modes. Using carefully controlled experimental conditions, we probed their conformational behavior and interactions with the high-affinity ligand quinine as a surrogate for cocaine. The ratios of bound and unbound aptamers in the mass spectra were used to rank them according to their apparent quinine-binding affinity, qualitatively matching the published ranking order. The arrival time differences between the free aptamer and aptamer-quinine complexes were consistent with a small ligand-induced conformational change, and found to inversely correlate with the affinity of binding. This mass spectrometry-based approach provides a fast and convenient way to study the molecular basis of aptamer-ligand recognition.

LC-MS/MS Analysis of Bile Acids in In Vitro Samples.

Over the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the quantification of bile acids (BA) and their conjugates in different matrices, such as plasma, blood, urine, and cell lysates. Numerous reports have indeed been published describing methods for quantitative determination of bile acids in plasma samples obtained during in vivo studies. However, information on bioanalytical methods suitable for determination of bile acids in in vitro samples remained scarce. Therefore, we presently report a simple and accurate LC-MS/MS method for the quantification of BA in cells (e.g., cultured human hepatocytes) and corresponding cell culture medium, obtained during in vitro experiments.

A generic approach for "shotgun" analysis of the soluble proteome of plant cell suspension cultures.

Cell suspension cultures from different plant species act as important model systems for studying cellular processes in plant biology and are often used as "green factories" for the production of valuable secondary metabolites and recombinant proteins. While mass spectrometry based proteome analysis techniques are ideally suited to study plant cell metabolism and other fundamental cellular processes from a birds eye perspective, they remain underused in plant studies. We describe a comprehensive sample preparation and multidimensional 'shotgun' proteomics strategy that can be generically applied to plant cell suspension cultures. This strategy was optimized and tested on an Arabidopsis thaliana ecotype Landsberg erecta culture. Furthermore, the implementation of strong cation exchange chromatography as a peptide fractionation step is elaborately tested. Its utility in mass spectrometry based proteome analysis is discussed. Using the presented analytical platform, over 13,000 unique peptides and 2640 proteins could be identified from a single plant cell suspension sample. Finally, the experimental setup is validated using Nicotiana tabacum cv. "Bright Yellow-2" (BY-2) plant cell suspension cultures, thereby demonstrating that the presented analytical platform can also be valuable tool in proteome analysis of non-genomic model systems.