Mapping protein structural changes by quantitative cross-linking.

Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.

Fabry disease: renal sphingolipid distribution in the α-Gal A knockout mouse model by mass spectrometric and immunohistochemical imaging.

Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis.

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.

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.

Scedosporium and Pseudallescheria low molecular weight metabolites revealed by database search.

The potential of mMass software search tool with new compound libraries was demonstrated on metabolomics of Scedosporium prolificans, S. apiospermum and Pseudallescheria boydii sensu stricto. Cyclic peptides pseudacyclins, small molecular weight tyroscherin analogues and various lipids were annotated by public software tool (http://www.mmass.org) utilising accurate matrix-assisted laser desorption/ionisation mass spectral data of intact fungal spores. Electrospray ionisation combined with tandem mass spectrometry was used for monohexosylceramide characterisation in fungal extracts.

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 user's guide, and video tutorials are freely available from www.mmass.org .

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.

Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins.

The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.

Deciphering Dorin M glycosylation by mass spectrometry.

The soft tick, Ornithodoros moubata, is a vector of several bacterial and viral pathogens including Borrelia duttoni, a causative agent of relapsing fever and African swine fever virus. Previously, a sialic acid-specific lectin Dorin M was isolated from its hemolymph. Here, we report on the complete characterization of the primary sequence of Dorin M. Using liquid chromatography coupled to mass spectrometry, we identified three different glycopeptides in the tryptic digest of Dorin M. The peptide, as well as the glycan part of all glycopeptides, were further fully sequenced by means of tandem mass spectrometry (MS2) and multiple-stage mass spectrometry (MS3). Two classical N-glycosylation sites were modified by high-mannose-type glycans containing up to nine mannose residues. The third site bore a glycan with four to five mannose residues and a deoxyhexose (fucose) attached to the proximal N-acetylglycosamine. The microheterogeneity at each site was estimated based on chromatographic behavior of different glycoforms. The fourth, a non-classical N-glycosylation site (Asn-Asn-Cys), was not glycosylated, probably due to the involvement of the cysteine residue in a disulfide bridge.

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.

In-situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing.

We report substantial in-situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide-coated matrix assisted laser desorption-ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium-tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in-vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI-Fourier transform ion cyclotron resonance (FTICR) and MALDI-time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption-ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results.

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.

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.

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization.

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibers is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.

Utilization of high-accuracy FTICR-MS data in protein quantitation experiments.

Human acute T-lymphoblastic leukemia cell line (CEM) treated with cisplatin, and the stable isotope labeling by amino acids in cell culture (SILAC) strategy were used to present an improved method of data processing in high-accuracy mass spectrometry (MS). By using peptide mass fingerprinting with low mass tolerance, we were able to utilize far more data retained in MS scans which would normally be missed by a standard processing method. This new way of data interpretation results in an improvement of the relevance of quantitation experiments and enabled us to search and quantify different types of posttranslational modifications. Furthermore, we used this technique to distinguish among different protein isoforms, commonly returned by Mascot search engine.

Tryptophan modification by 2-hydroxy-5-nitrobenzyl bromide studied by MALDI-TOF mass spectrometry.

The reaction with 2-hydroxy-5-nitrobenzyl bromide (HNB) is a common covalent modification of tryptophan. It results in several products which have been described by classical physico-chemical methods. To improve the understanding of the HNB-modified tryptophan structure, we synthesized a model peptide containing one tryptophan only, modified it by HNB, and analyzed the product by MALDI-TOF mass spectrometry. Surprisingly, several multi-modified products (up to 5 HNB moieties per one tryptophan) were identified. the influence of HNB concentration and pH on the degree of modification was also analyzed. In addition, a splitting of modified tryptophan peaks in MALDI-TOF spectrum was described; most probably, this effect is a common MALDI artifact of nitro-aromatic compounds which facilitates the identification of HNB-modified tryptophan by MALDI-TOF MS significantly.

Citraconylation--a simple method for high protein sequence coverage in MALDI-TOF mass spectrometry.

Lysine epsilon -amino group reacts with citraconic anhydride forming a derivative, which is stable on terms for trypsin cleavage. This modification changes the spectrum of peptides formed by the trypsin action; as the number of trypsin-sensitive sites is reduced, the peptides with higher molecular mass can survive in the digest. The various studies of proteins by MALDI-TOF mass spectrometry are often complicated by the low sequence coverage of the peptide chain. This paper demonstrates that the modification of proteins by citraconylation before trypsin cleavage represents a simple experimental technique, which allows a significant increase of sequence coverage in MALDI-TOF mass spectrometry. This improvement is caused both by change of trypsin fragmentation pattern and by disturbance of the protein's native tertiary structure.

Analysis of tryptophan surface accessibility in proteins by MALDI-TOF mass spectrometry.

Surface accessible amino acids can play an important role in proteins. They can participate in enzyme's active center structure or in specific intermolecular interactions. Thus, the information about selected amino acids' surface accessibility can contribute to the understanding of protein structure and function. In this paper, we present a simple method for surface accessibility mapping of tryptophan side chains by their chemical modification and identification by MALDI-TOF mass spectrometry. The reaction with 2-hydroxy-5-nitrobenzyl bromide, a common and highly specific covalent modification of tryptophan, seems to be very useful for this purpose. The method was tested on four model proteins with known spatial structure. In the native proteins (1) only surface accessible tryptophan side chains were found to react with the modification agent and (2) no buried one was found to react at lower reagent concentrations. These results indicate that the described method can be a potent tool for identification of surface-located tryptophan side chain in a protein of unknown conformation.