Martin Strohalm

mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data.

While tools for the automated analysis of MS and LC-MS/MS data are continuously improving, it is still often the case that at the end of an experiment, the mass spectrometrist will spend time carefully examining individual spectra. Current software support is mostly provided only by the instrument vendors, and the available software tools are often instrument-dependent. Here we present a new generation of mMass, a cross-platform environment for the precise analysis of individual mass spectra. The software covers a wide range of processing tasks such as import from various data formats, smoothing, baseline correction, peak picking, deisotoping, charge determination, and recalibration. Functions presented in the earlier versions such as in silico digestion and fragmentation were redesigned and improved. In addition to Mascot, an interface for ProFound has been implemented. A specific tool is available for isotopic pattern modeling to enable precise data validation. The largest available lipid database (from the LIPID MAPS Consortium) has been incorporated and together with the new compound search tool lipids can be rapidly identified. In addition, the user can define custom libraries of compounds and use them analogously. The new version of mMass is based on a stand-alone Python library, which provides the basic functionality for data processing and interpretation. This library can serve as a good starting point for other developers in their projects. Binary distributions of mMass, its source code, a detailed users guide, and video tutorials are freely available from www.mmass.org .

mMass data miner: an open source alternative for mass spectrometric data analysis.

Go to www.mmass.org and download the appropriate version of mMass for your computer. Versions are available for Linux, Windows, and Mac OSX. File à Open mMass will open its own files (.msd) and ASCII files (.txt). When you open an ASCII (.txt) file, save it as (save as) a .msd file. Control-click (right-click) the cursor over an area of the spectrum to enlarge that area. Double-click on the spectrum to return to the full spectrum. Tools à Label Peak : Cursor is now labeled with a small green +. Pull the cursor across the top of a peak, and a label appears on the spectrum and in a list to the right of the spectrum. To delete a label, highlight it in the peak list, and hit delete. View à Canvas Properties m/z precision, intensity precision, bars height, gel height, canvas font size, label font size, notation length Tools à Mass Calculator à Isotope Pattern window File à Export à Export Spectrum Image Choose size, resolution, font scale, line scale, format (PNG, JPEG, or TIFF). To change color of spectrum: control-click (right-click) on the colored dot for that spectrum à colour Other: View à Peak Labels à vertical/horizontal View à Peak List à lots of choices Sequence à New à Sequence Editor (This is all about peptides and proteins.) Processing à Peak Picking à wrench button Check and see if your data fits any of the types listed. Processing means treating a batch of spectra all the same way. If you use images generated with mMass in publications, reports or posters, be sure to say you used mMass and cite it. mMass 3: A Cross-Platform Software Environment for Precise Analysis of Mass Spectrometric Data Strohalm M, Kavan D, Novák P, Volný M, Havlíček V Anal Chem 82 (11), 4648-4651 (2010). mMass Data Miner: an Open Source Alternative for Mass Spectrometric Data Analysis Strohalm M, Hassman M, Košata B, Kodíček M Rapid Commun Mass Spec 22 (6), 905-908 (2008).

mMass as a Software Tool for the Annotation of Cyclic Peptide Tandem Mass Spectra

Natural or synthetic cyclic peptides often possess pronounced bioactivity. Their mass spectrometric characterization is difficult due to the predominant occurrence of non-proteinogenic monomers and the complex fragmentation patterns observed. Even though several software tools for cyclic peptide tandem mass spectra annotation have been published, these tools are still unable to annotate a majority of the signals observed in experimentally obtained mass spectra. They are thus not suitable for extensive mass spectrometric characterization of these compounds. This lack of advanced and user-friendly software tools has motivated us to extend the fragmentation module of a freely available open-source software, mMass (http://www.mmass.org), to allow for cyclic peptide tandem mass spectra annotation and interpretation. The resulting software has been tested on several cyanobacterial and other naturally occurring peptides. It has been found to be superior to other currently available tools concerning both usability and annotation extensiveness. Thus it is highly useful for accelerating the structure confirmation and elucidation of cyclic as well as linear peptides and depsipeptides.

Laser Desorption-Ionization of Lipid Transfers: Tissue Mass Spectrometry Imaging without MALDI Matrix

Mass spectrometry imaging of tissue-lipid transfers without MALDI matrix is demonstrated. Commercially available nanostructured surfaces (nano-assisted laser desorption-ionization or NALDI) are used as substrates for imprinting of tissue sections. The lithographic transfers are then washed and the two-dimensional distribution of the lipids is imaged by laser desorption-ionization mass spectrometry. The NALDI imaging of lipid transfers is compared with standard MALDI imaging of matrix-coated tissue sections. The obtained images are of the same quality, and no spatial information is lost due to the imprinting process. NALDI imaging is faster due to the absence of the time-consuming matrix deposition step, and the NALDI mass spectra are less complex and easier to interpret than standard MALDI. In this particular application example, NALDI mass spectrometry is able to identify the same lipid species as MALDI mass spectrometry and provides better distinction between kidney and adrenal gland tissues based on the lipid analysis.

Molecular mass spectrometry imaging in biomedical and life science research

This review describes the current state of mass spectrometry imaging (MSI) in life sciences. A brief overview of mass spectrometry principles is presented followed by a thorough introduction to the MSI workflows, principles and areas of application. Three major desorption-ionization techniques used in MSI, namely, secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) are described, and biomedical and life science imaging applications of each ionization technique are reviewed. A separate section is devoted to data handling and current challenges and future perspectives are briefly discussed at the end.

Spectral analysis of doxorubicin accumulation and the indirect quantification of its DNA intercalation.

There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.

Poly[N-(2-hydroxypropyl)methacrylamide]-based tissue-embedding medium compatible with MALDI mass spectrometry imaging experiments.

Traditional tissue-sectioning techniques for histological samples utilize various embedding media to stabilize the tissue on a sectioning target and to provide a smooth cutting surface. Due to the ion suppression effect in MALDI ionization and number of background peaks in the low-mass region, these media are not suitable for mass spectrometry imaging (MSI) experiments. To overcome this, droplets of water are often used to mount the tissue on a sectioning target, but the ice block formed around the tissue does not provide a good support for sectioning of fragile samples. In this work, we propose a novel embedding media, compatible with MALDI ionization and MSI experiments, based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Using a reversible addition-fragmentation chain transfer polymerization technique, well-defined pHPMA polymer with narrow mass distribution was prepared. Benefits of the resulted pHPMA-based embedding media were tested on different tissue samples.

Plant-microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil.

During the second half of the last century a large amount of substances toxic for higher organisms was released to the environment. Physicochemical methods of pollutant removal are difficult and prohibitively expensive. Using biological systems such as microorganisms, plants, or consortia microorganisms-plants is easier, cheaper, and more environmentally friendly. The aim of this study was to isolate, characterize and identify microorganisms from contaminated soil and to find out the effect of plants on microbial diversity in the environment. Microorganisms were isolated by two approaches with the aim to find all cultivable species and those able to utilise biphenyl as a sole source of carbon and energy. The first approach was direct extraction and the second was isolation of bacteria after enrichment cultivation with biphenyl. Isolates were biochemically characterized by NEFERMtest 24 and then the composition of ribosomal proteins in bacterial cells was determined by MALDI-TOF mass spectrometry. Ribosomal proteins can be used as phylogenetic markers and thus MALDI-TOF MS can be exploited also for taxonomic identification because the constitution of ribosomal proteins in bacterial cells is specific for each bacterial species. Identification of microorganisms using this method is performed with the help of database Bruker Daltonics MALDI BioTyper. Isolated bacteria were analyzed from the point of the bphA gene presence. Bacteria with detected bphA gene were then taxonomically identified by 16S rRNA sequence. The ability of two different plant species, tobacco (Nicotiana tabacum) and nightshade (Solanum nigrum), to accumulate PCBs was studied as well. It was determined that various plant species differ in the PCBs accumulation from the contaminated soil. Also the content of PCBs in various plant tissues was compared. PCBs were detected in roots and aboveground biomass including leaves and berries.

Spatial Distribution of Glycerophospholipids in the Ocular Lens

Knowledge of the spatial distribution of lipids in the intraocular lens is important for understanding the physiology and biochemistry of this unique tissue and for gaining a better insight into the mechanisms underlying diseases of the lens. Following our previous study showing the spatial distribution of sphingolipids in the porcine lens, the current study used ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) to provide the whole lipidome of porcine lens and these studies were supplemented by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) of the lens using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to determine the spatial distribution of glycerophospholipids. Altogether 172 lipid species were identified with high confidence and their concentration was determined. Sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines were the most abundant lipid classes. We then determined the spatial and concentration-dependent distributions of 20 phosphatidylcholines, 6 phosphatidylethanolamines, and 4 phosphatidic acids. Based on the planar molecular images of the lipids, we report the organization of fiber cell membranes within the ocular lens and suggest roles for these lipids in normal and diseased lenses.

Time-dependent oxidation during nano-assisted laser desorption ionization mass spectrometry: a useful tool for structure determination or a source of possible confusion?

This work reports on a new and extremely simple approach for determination of a double bond position by a laser desorption ionization mass spectrometry. It is solely based on the catalytic properties of nanostructured surfaces used in nanoassisted laser desorption ionization experiments. These surfaces can induce oxidation of analytes, which results in a mass shift that can be detected by mass spectrometry. If a site of unsaturation is oxidized and cleaved, the m/z difference is diagnostic of the position of a double bond. By demonstrating that the oxidation depends on the analyte surface dwell time, it was proven that it is caused by the surface activity and not by the laser desorption ionization process itself. Control matrix-assisted laser desorption/ionization (MALDI) experiment showed only a limited partial oxidation and no time dependency of the process. The ability to determine a position of a double bond was demonstrated on polyunsaturated phospholipids and cyclosporine A. In some other cases, however, the unexpected oxidation could cause confusion, as demonstrated for a glycosphingolipid from a porcine brain extract.