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Rationale: Selective derivatization of peptide N-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/−) under charge reduction conditions. Overlapped positive and negative tandem mass spectra show b-ions making the assignment of b-ion series fragments easy and accurate.// Methods: We developed an FBSA–peptide microwave-assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non-tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high-quality data set of sulfonated b-ions obtained in negative tandem mass spectra of singly charged FBSA–peptides were matched to detected b-ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y-ions in positive tandem mass spectra to identify complete peptide sequences.// Results: The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high-intensity signals of b- and y-ions) compared to commonly used N-terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b-ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive- and negative ion modes, respectively. High visibility of b- ion series in negative ion mode can be attributed to N-terminal sulfonation that had no negative effect on the production of b- and y-ion series in positive ion mode.// Conclusions: The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b-ions in positive- and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.

RATIONALE: Selective derivatization of peptide N-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables chemically activated fragmentation in ESI+/- (positive and negative ion mode) under charge reduction conditions. Overlapped positive and negative tandem mass spectra pinpoint b-ions making the assignment of b-ion series fragments easy and accurate. METHODS: We developed a FBSA-peptide microwave assisted derivatization procedure. Derivatized and non-derivatized bovine serum albumin tryptic peptides and insulin non-tryptic peptide were compared after MS/MS analysis in positive and negative ion mode. High-quality dataset of sulfonated b-ions obtained in negative tandem mass spectra of singly charged FBSA-peptides were matched to detected b-ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y-ions in positive tandem mass spectra to identify complete peptide sequences. RESULTS: The FBSA derivatization procedure produced a significantly improved MS/MS dataset (populated by high-intensity signals of b- and y-ions) in comparison to commonly used N-terminal sulfonation reagents. Undesired side reactions do not occur and the procedure reduces the derivatization time. It was found that b-ions comprise 15% and 13% of all fragment ions generated in positive and negative ion mode, respectively. High visibility of b-ion series in negative ion mode can be attributed to the N-terminal sulfonation which had no effect on the production of b-ion series in positive ion mode. CONCLUSIONS: The FBSA derivatization and de novo sequencing approach outlined here provides a reliable method for accurate peptide sequence assignment. Increased production of the b-ions in positive and negative ion mode greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.

Aims Adenoviruses that have C NGR C VSG C AGR C peptide inserted into fiber (AdFNGR) or hexon (AdHNGR) protein, respectively, showed increased transduction of endothelial cells. In this study we investigated if cysteines within the C NGR C VSG C AGR C sequence inserted into Ad5 fiber or hexon protein form disulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR. Methods Transduction efficiency of adenoviruses was done by counting infected cells under the microscope. Adenovirus attachment and internalization were measured by qPCR. Flow cytometry was used to evaluate the expression of CD13 and integrins. Gene knockdown was achieved by transfection of small interfering RNA. Mass spectrometry was used for determining disulfide bonds in adenovirus fiber and hexon protein. Molecular modeling was use to predict interaction of CNGRCVSGCAGRC peptide and CD13. Key findings AdFNGR and AdHNGR attach better to CD13 and/or αvβ3 integrin-positive cells than Adwt. Reducing disulfide bonds using DTT decreased transduction efficiency and attachment of both AdFNGR and AdHNGR. Cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds. Knockdown of αvβ3 integrin reduced increased transduction efficiency of both AdFNGR and AdHNGR, while CD13 knockdown had no effect, indicating that retargeting properties of these viruses rely mainly on αvβ3 integrin expression. Significance Insertion site of NGR-containing peptides as well as NGR flanking residues are critical for receptor binding affinity/specificity and transduction efficiency of NGR retargeted adenoviral vectors.

Widespread application of silver nanoparticles (AgNPs), due to their antibacterial and antifungal properties, increases their release into the environment and potential detrimental impact on living organisms. Plants may serve as a potential pathway for AgNPs bioaccumulation and a route into the food chain, hence investigation of AgNP phytotoxic effects are of particular importance. Since proteins are directly involved in stress response, studies of their abundance changes can help elucidate the mechanism of the AgNP-mediated phytotoxicity. In this study, we investigated proteomic changes in tobacco (Nicotiana tabacum) exposed to AgNPs and ionic silver (AgNO3). A high overlap of differently abundant proteins was found in root after exposure to both treatments, while in leaf, almost a half of the proteins exhibited different abundance level between treatments, indicating tissue-specific responses. Majority of the identified proteins were down-regulated in both tissues after exposure to either AgNPs or AgNO3; in roots, the most affected proteins were those involved in response to abiotic and biotic stimuli and oxidative stress, while in leaf, both treatments had the most prominent effect on photosynthesis-related proteins. However, since AgNPs induced higher suppression of protein abundance than AgNO3, we conclude that AgNP effects can, at least partially, be attributed to nanoparticle form.

Metabolic syndrome, characterized by obesity, low-grade inflammation, insulin resistance, hyperglycemia, dyslipidemia and hypertension, is a major risk factor for cardiovascular mortality. Preclinical studies on recently discovered classes of lipids – fatty acid esters of hydroxy fatty acids (FAHFA) have revealed their anti-inflammatory and insulin- sensitizing potential. The FAHFA levels are significantly decreased in insulin-resistant individuals, their application exhibited anti- inflammatory effects and restoring the glucose- insulin homeostasis. The aim of our research was to analyze the overall FAHFA composition in a common diet, as only a partial FAHFA composition has been revealed so far (only the PAHSA subclass was analyzed in a few foods). A new approach to the FAHFAs analysis includes nano-LC and post-column modifier followed by negative ion mass spectrometry, in order to obtain maximum sensitivity. Analysis of different foods – oat (whole grain, coarse flakes and fine flakes), apple, clementine, lemon, strawberry, blueberry, mango, kiwi, avocado, pineapple, banana, onion, garlic, cherry tomato, carrot, parsley root, pepper and radish – exhibited wide inter-food variation in the FAHFA profiles. Sixteen analyzed FAHFAs (palmitic, oleic, palmitoleic and stearic hydroxy-esters) showed microgram to low nanogram levels (0.165 ng/g – 32 μg/g FW), with the highest abundancy in oat, clementine, garlic and pineapple. Stearic acid hydroxy stearic acid (SAHSA) was the most abundant FAHFA, especially in the food with antioxidative, anti-inflammatory and beneficial metabolic effects. In contrary, the PAHSA - previously proven to have the strongest antihyperglycemic and insulin-sensitizing effects, was not present in some foods (radish, avocado, mango, lemon, cherry tomato, kiwi). Our study proves the importance of overall FAHFA analysis in food (especially in a functional food), because of their potential metabolic benefits and possible future incorporation in special diets.

Vipera ammodytes (Va), is the European venomous snake of the greatest medical importance. We analyzed whole venom proteome of the subspecies V. ammodytes ammodytes (Vaa) from Serbia for the first time using the shotgun proteomics approach and identified 99 proteins belonging to four enzymatic families: serine protease (SVSPs), L-amino acid oxidase (LAAOs), metalloproteinases (SVMPs), group II phospholipase (PLA2s), and five nonenzymatic families: cysteine- rich secretory proteins (CRISPs), C-type lectins (snaclecs), growth factors -nerve (NGFs) and vascular endothelium (VEGFs), and Kunitz-type protease inhibitors (SPIs). Considerable enzymatic activity of LAAO, SVSPs, and SVMPs and a high acidic PLA2 activity was measured implying potential of Vaa to produce haemotoxic, myotoxic, neuro and cardiotoxic effects. Moreover, significant antimicrobial activity of Vaa venom against Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was found. The crude venom shows considerable potential cytotoxic activity on the C6 and HL60 and a moderate level of potency on B16 cell lines. HeLa cells showed the same sensitivity, while DU 145 and PC-3 are less sensitive than as normal cell line. Our data demonstrated a high complexity of Vaa and considerable enzymatic, antibacterial and cytotoxic activity, implying a great medical potential of Vaa venom as a promising source for new antibacterial and cytostatic agents.

Background Reliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample. Methods In total, 16 urine samples having more than 105 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested. Results Here we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing. Conclusions Our findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.

RATIONALE One of the most challenging tasks of proteomics is peptide de novo sequencing. 4- Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high- quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non-specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in high-throughput de novo sequencing. METHODS We developed a SPITC peptide derivatization procedure under acidic conditions (pH ≤5). Derivatized peptides were analyzed by matrix- assisted laser desorption/ionization (MALDI-MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high-throughput peptide de novo sequencing. This high-throughput peptide de novo sequencing was tested on Fusarium delphinoides, an organism with an uncharacterized genome. RESULTS The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over-sulfonation effect measured on lysine- containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. CONCLUSIONS The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high-throughput peptide de novo sequencing. High-throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non-sequenced genomes.

Brettanomyces bruxellensis is one of the most important spoilage yeasts in red wine production. The aim of this paper was to investigate the diversity of B. bruxellensis isolates in regard to their proteomic, growth, and metabolic profiles. Ten isolates were obtained from several wineries in Croatian winegrowing regions during different phases of wine production. Proteomic analysis revealed 12 proteins that were expressed by all tested isolates and the reference strain. These proteins could be used as biomarkers in Dekkera/B. bruxellensis yeast identification. Five of these proteins were involved in carbohydrate metabolism (enolase, hxk2p, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and pyruvate decarboxylase), and four were involved in protein biosynthesis (elongation factor 1-alpha, eukaryotic translation initiation factor 5a, 60s ribosomal protein 13, and putative cytosolic ribosomal protein s24). One each was involved in cellular stress responses to glucose starvation (heat shock protein ssb1); ubiquitin conjugation pathways such as transcription, proteolysis trafficking, and kinase activation (ubiquitin); or nitrogen metabolism (peptidyl-prolyl cis-trans isomerase). Isolates identified as B. bruxellensis and taken during malolactic fermentation expressed 18 similar proteins, and isolates from aging in steel vessels/wood barrels or bottled wine expressed 23 and 24 similar proteins, respectively. Growth and metabolic profiles of these isolates were evaluated in two growth media (glucose complex and wine-mimicking media). The growth profiles of the tested isolates and conversion of hydroxycinnamic acids varied among the investigated media in that the use of glucose complex medium resulted in faster growth and consumption of hydroxycinnamic acids and in higher production of volatile phenols and esters. The results obtained suggest the possibility of applying proteomic fingerprinting for the identification and differentiation of B. bruxellensis wine isolates and reveal different spoilage capacities, such as growth and metabolic profiles, of the tested isolates.

The photophysical properties and photochemical reactivities of a series of bis-naphthols 4a–4e and bis-anthrols 5a and 5e were investigated by preparative irradiation in CH3OH, fluorescence spectroscopy and laser flash photolysis (LFP). Methanolysis taking place via photodehydration (bis-naphthols: ΦR = 0.04–0.05) is in competition with symmetry breaking charge separation (SB-CS). The SB-CS gave rise to radical ions that were detected for 4a and 4e by LFP. Photodehydration gave quinone methides (QMs) that were also detected by LFP (λmax = 350 nm, τ ≈ 1–2 ms). In the aqueous solvent, excited state proton transfer (ESPT) competes with the abovementioned processes, giving rise to naphtholates, but the process is inefficient and can only be observed in the buffered aqueous solution at pH > 7. Since the dehydration of bis-naphthols delivers QMs, their potential antiproliferative activity was investigated by an MTT test on three human cancer cell lines (NCI-H1299, lung carcinoma; MCF-7, breast adenocarcinoma; and SUM159, pleomorphic breast carcinoma). Cells were treated with 4 or 5 with or without irradiation (350 nm). An enhancement of the activity (up to 10-fold) was observed upon irradiation, which may be associated with QM formation. However, these QMs do not cross-link DNA. The activity is most likely associated with the alkylation of proteins present in the cell cytoplasm, as evidenced by photoinduced alkylation of bovine and human serum albumins by 4a.

Separation of plant proteins by means of electrophoretic techniques is quite challenging since different compounds typical for plant cells can interfere and/or reduce the effectiveness of the protein isolation. This is particularly problematic for two-dimensional electrophoresis (2-DE). Therefore, it is important to optimize protein extraction and to establish a robust protocol for 2-DE and downstream processing, primarily mass spectrometry (MS) analysis. Here we give a detailed protocol for protein extraction using phenol method, 2-DE, and MALDI-MS analysis.

Binding of three ruthenium(ii) compounds of general formula mer-[Ru(L3)(N-N)X][Y] (where L3 = 4-chloro-2,2:6,2-terpyridine (Cl-tpy); N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach) or 2,2-bipyridine (bipy); X = Cl; Y = Cl) to human serum albumin (HSA) has been investigated by nano-LC/nano-ESI MS and docking studies. A bottom-up proteomics approach has been applied for the structural characterization of metallated proteins and the data were analyzed in both the positive and negative ion mode. The negative ion mode was achieved after the post-column addition of an isopropanol solution of formaldehyde that enabled sample ionization at micro-flow rates. The negative ion mode MS has been proved to be beneficial for the analysis of binding sites on ruthenated protein in terms of ion charge reduction and consequent simplification of target sequence identification based on isotopic differences between ruthenated and non-ruthenated peptides. Moreover, the negative ion mode ESI MS shows the advantage of singly charged ion formation and, unlike MALDI MS, it does not cause complete ligand fragmentation, merging the benefits of each method into a single experiment. Six target sequences were identified for the binding of en and dach compounds, and four sequences for the binding of bipy. All compounds have been found to bind histidine and one aspartate residue. Docking studies showed that the identified sequences are the constituents of five distinct binding sites for en and dach, or two sites for the bipy complex. The selection of binding sites seems to be dependent on the chelate ligand and the form of the complex prior or after hydrolysis of the leaving chloride ligand. This is a pre-copyedited, author-produced version of an article accepted for publication in Metallomics following peer review. The version of record Nišavić, M.; Janjić, G. V.; Hozić, A.; Petković, M.; Milčić, M. K.; Vujčić, Z.; Cindrić, M. Positive and Negative Nano-Electrospray Mass Spectrometry of Ruthenated Serum Albumin Supported by Docking Studies: An Integrated Approach towards Defining Metallodrug Binding Sites on Proteins. Metallomics 2018, 10 (4), 587–594 is available online at: [https://doi.org/10.1039/c7mt00330g]

Salt and drought stress are important abiotic factors that negatively affect plant growth and productivity. Defense mechanisms, which plants have developed to cope with stress, are followed by alterations in a genome expression profile that in turn result in qualitative and quantitative change of the proteome. Although proteomic-based approach for studies of plant responses to salinity and drought has already been successfully employed in several plants, for cactus species such analyses have not been done so far. Therefore, in this study we have performed proteomic analysis of Mammillaria gracilis Pfeiff. in vitro grown cultures, callus and tumor, exposed to iso-osmotic NaCl and mannitol. Obtained results differed among analyzed tissues. The higher number of differentially expressed proteins after either salt or mannitol treatment was revealed in tumor compared to callus. According to classification to different functional categories, majority of the identified callus responsive proteins belongs to protein synthesis and processing category, while the highest number of identified tumor proteins belongs to category of metabolism, which suggest that the mechanisms that mediate responses to salt- and mannitol-induced stress in cactus callus and tumor are dependent on tissue type. Down-regulation of proteins involved in cell protection suggests the inability of tumor to activate protective processes against salinity and osmotic stress.

Since silver nanoparticles (AgNPs) are a dominant nanomaterial in consumer products, there is growing concern about their impact on the environment. Although numerous studies on the effects of AgNPs on living organisms have been conducted, the interaction of AgNPs with plants has not been fully clarified. To reveal the plant mechanisms activated after exposure to AgNPs and to differentiate between effects specific to nanoparticles and ionic silver, we investigated the physiological, ultrastructural and proteomic changes in seedlings of tobacco (Nicotiana tabacum) exposed to commercial AgNPs and ionic silver (AgNO3) from the seed stage. A higher Ag content was measured in seedlings exposed to AgNPs than in those exposed to the same concentration of AgNO3. However, the results on oxidative stress parameters obtained revealed that, in general, higher toxicity was recorded in AgNO3-treated seedlings than in those exposed to nanosilver. Ultrastructural analysis of root cells confirmed the presence of silver in the form of nanoparticles, which may explain the lower toxicity of AgNPs. However, the ultrastructural changes of chloroplasts as well as proteomic study showed that both AgNPs and AgNO3 can affect photosynthesis. Moreover, the majority of the proteins involved in the primary metabolism were up-regulated after both types of treatments, indicating that enhanced energy production, which can be used to reinforce defensive mechanisms, enables plants to cope with silver-induced toxicity.

Separation of plant proteins by means of electrophoretic techniques is quite challenging since different compounds typical for plant cells can interfere and/or reduce the effectiveness of the protein isolation. This is particularly problematic for two-dimensional electrophoresis (2-DE). Therefore, it is important to optimize protein extraction and to establish a robust protocol for 2-DE and downstream processing, primarily mass spectrometry (MS) analysis. Here we give a detailed protocol for protein extraction using phenol method, 2-DE, and MALDI-MS analysis.

In this work, a simple way for study the possibility of formation a vapor cluster species of tetrachloroauric acid (HAuCl4), using the laser ablation in the absence of a buffer or reactive atmosphere, and without a postablation supersonic expansion on a commercial matrix assisted laser desorption/ionization time-of-flight mass spectrometer, is reported. Tetrachloroauric acid is known as precursor for the synthesis of gold nanostructures and the complex salts; therefore it is an important task to discover and quantify the species arising from HAuCl4, in order to understand their role in the gold assisted reactions. Mass spectrum of HAuCl4 in a reflector negative-ion mode contains the hydrated mono- and dinuclear gold clusters in the m/z range 286–436, and gold chloride clusters in the m/z range 447–795. In the first part of spectrum, m/z range 286–436, the hydrated gold cluster species of type Au n − (H2O)m (n = 1–2; m = 1, 2, 5, 7, 8) and [Aun(OH)k]−(H2O)m (n = 1–2; k = 1–2; m = 1, 4–8) were found. Besides that, there are gold chloride clusters with general formula [AuHr(HCl)2]−(H2O)m (m = 1–5; 8–9; r = 0–2) in this part of spectrum. In the second part of spectrum, the m/z range 447–795, only gold chloride clusters were obtained. Their general formulae can be written as [AuClt(HCl)v]−(H2O)m (t = 1–4; v = 5–8; m = 2–4, 6–8) and [Aun(HCl)v]−(H2O)m (n = 1–2, v = 4–5, m = 1–2, 5, 7). The analysis of concentration effects on the LDI mass spectra of gold clusters reveals that the relative intensities of signals for the mono- and dinuclear Au clusters increase with decreasing the concentration of water HAuCl4 solutions.

RATIONALE We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation. METHODS The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation. RESULTS The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells. CONCLUSIONS Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article. Copyright © 2013 John Wiley & Sons, Ltd. Copyright

Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile- phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2, 2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.

Background The αv-integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie and adenovirus receptor. However, until now, it has been unclear whether disulfide bonds in the RGD4C motif influence the retargeting potential of RGD4C-modified Ad5. Methods Replication deficient Ad5 bearing wild-type fiber (Ad5wt) or RGD4G, RGD4C and RGD2C2G insertions within the HI loop of the fiber protein (Ad5RGD4G, Ad5RGD4C and Ad5RGD2C2G, respectively) were used to transduce a panel of cancer cell lines, with or without previous treatment of these Ad5s with the reducing agent dithiothreitol (DTT). In parallel, native and DTT-treated fiber proteins isolated from purified Ad5RGD4C were compared by mass spectrometry. Results Ad5RGD4C transduced all studied cell lines much more efficiently than Ad5wt, whereas Ad5RGD4G transduced cells only slightly more efficiently than Ad5wt. DTT treatment had no effect on cell transduction by wild-type Ad5wt and Ad5RGD4G but abolished the increased transduction efficacy of Ad5RGD4C in a dose-dependent manner. The mass spectra of native and DTT-reduced tryptic digests of the Ad5RGD4C fiber protein are consistent with the presence of a C547–C549 linkage in the C547DC549RGDC553FC555 motif. Finally, the high transduction efficacy of Ad5RGD4C is conserved in Ad5RGD2C2G. Conclusions We provide genetic and biochemical data strongly suggesting that cysteines C547 and C549 from the C547DC549RGDC553FC555 motif inserted in the HI loop of the Ad5 fiber form a single disulfide bond, with this disulfide bond being crucial for Ad5RGD4C retargeting to av-integrins. Copyright © 2012 John Wiley & Sons, Ltd.

S-Adenosylhomocysteine hydrolase (AHCY) deficiency is a rare congenital disorder in methionine metabolism clinically characterized by white matter atrophy, delayed myelination, slowly progressive myopathy, retarded psychomotor development and mildly active chronic hepatitis. In the present study, we utilized a comparative proteomics strategy based on 2-DE/MALDI-MS and LC/ESI-MS to analyze plasma proteins from three AHCY-deficient patients prior to and after receiving dietary treatment designed to alleviate disease symptoms. Obtained results revealed candidate biomarkers for the detection of myopathy specifically associated with AHCY deficiency, such as carbonic anhydrase 3, creatine kinase, and thrombospondin 4. Several proteins mediating T-cell activation and function were identified as well, including attractin and diacylglycerol kinase α. Further validation and functional analysis of identified proteins with clinical value would ensure that these biomarkers make their way into routine diagnosis and management of AHCY deficiency.

A rapid and sensitive reversed phase liquid chromatography- tandem mass spectrometry (LC-MS-MS) method is developed for the determination of doxazosin in canine plasma. The samples are prepared by precipitation of proteins using a mixture of methanol and acetonitrile, followed by freezing and evaporation of the organic solvent. The remaining dry residue is redissolved in mobile phase and analyzed by LC-MS-MS with positive electrospray ionization using the selected reactions monitoring mode. An XTerra MS C(18) column, a mobile phase composed of acetonitrile and 2mM ammonium acetate with gradient elution, and a flow rate of 400 microL/min are employed. The elution times for prazosin (internal standard) and doxazosin are approximately 8 and 10 min, respectively. Calibration curves are linear in the 1-20 ng/mL concentration range. Limits of detection and quantification are 0.4 ng/mL and 1.2 ng/mL, respectively. Recovery is higher than 94%. Intra- and inter-day relative standard deviations are below 7% and 8%, respectively. The method is applied for the determination of doxazosin plasma levels following a single administration of doxazosin base and doxazosin mesylate tablets (2 mg dose) to dogs in the fed state. The results indicate possible superiority of the mesylate salt on the plasma input rates of doxazosin.

Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4 Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4 Cl-tpy)(dach)Cl](+) (2) (where 4 Cl-tpy = 4-chloro-2,2:6,2 -terpyridine, en = 1,2-diaminoethane and dach = 1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77 +/- 0.14 and 6.95 +/- 0.43 mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue. (C) 2016 Elsevier Inc. All rights reserved.

For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonas and Stenotrophomonas genera, considering acquired resistance of Pseudomonas species, as well as the intrinsic resistance of Stenotrophomonas species. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF. The results of the standard PCR test, 16S rRNA gene sequencing and MALDI-TOF analysis confirmed 35 strains to belong to the Pseudomonas genus. Standard PCR test and VITEK MS device confirmed that 10 strains belong to Stenotrophomonas maltophilia species. Three strains were positive in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Three strains were negative in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Although modern test methods that have very high specificity (PCR, 16S rRNA gene Pseudomonas and Stenotrophomonas species for 6 isolates could not be reached using the above mentioned methods. S obzirom na značaj stečene rezistencije Pseudomonas vrsta, kao i na intrinzičnu rezistenciju Stenotrophomonas vrsta, a u cilju preciznog ispitivanja osetljivosti na antibiotike, neophodna je jasna diferencijacija pripadnika ovih rodova bakterija. U tom cilju su u identifikaciji 51 izolata poreklom od riba korišćene metode: standardni PCR, 16S rRNA sekvenciranje gena, MALDI-TOF. Rezultati standardnog PCR testa, 16S rRNA sekvenciranja gena i MALDI-TOF analize su za 35 sojeva potvrdili pripadnost rodu Pseudomonas. Standardnim PCR testom i primenom aparata VITEK MS utvrđeno je da 10 sojeva pripada vrsti Stenotrophomonas maltophilia. U 16S rRNA sekvenciranju gena 3 soja koja su bila pozitivna u oba standardna PCR testa identifikovana su kao 99% Pseudomonas sp. i 99% Stenotrophomonas sp. VITEK MS je ova tri soja u prvoj identifikaciji identifikovao kao 99% Stenotrophomonas, a u ponovljenoj identifikaciji kao 99% Pseudomonas. Ti sojevi su na aparatu MALDI TOF/TOF 4800 Plus bili identifikovani kao Stenotrophomonas. U 16S rRNA sekvenciranju gena tri soja koja su bila negativna u oba standardna PCR testa su identifikovana kao 99% Pseudomonas sp. i 99% Stenotrophomonas sp. Aparat VITEK MS je ova tri soja identifikovao u jednoj identifikaciji kao 99% Stenotrophomonas, a u drugoj identifikaciji 99% kao Pseudomonas. Ti sojevi su na aparatu MALDI TOF/ TOF 4800 Plus bili identifikovani kao Stenotrophomonas. Iako su u ovom istraživanju korišćene savremene metode ispitivanja koje imaju vrlo visoku specifičnost (PCR, 16s rRNK sequencing, MALDI TOF) precizna diferencijacija Pseudomonas i Stenotrophomonas vrsta nije mogla biti postignuta.

Mass spectrometry structural characterization is an essential tool in validating the quality of PEG-rHu-proteins. However, in either case top-down or bottom-up fashion, the interference of high intensity PEG signals on MS detection or detrimental influence of PEG on protein structure, leads to incomplete structural characterization. We propose here a method that permits complete and reliable structural characterization of PEGylated recombinant human granulocyte-colony stimulating factor (rHuG-CSF). The approach includes on-column 2-methoxy-4, 5-dihydro-1H-imidazole derivatization of digested PEG rHuG-CSF and subsequent LC/MS investigation. By comparing the LC/MS retention of derivatized and underivatized digested PEG rHuG-CSF, location of the PEG attachment within rHuG-CSF could be deduced. Besides, the protein sequence coverage and position of the disulfide bridges was fully deducible from the MS data interpretation. Additionally, ultra performance liquid chromatography– mass spectrometry-to-the-E (UPLC– MSE) was introduced for analysis of label-free digested PEG rHuG-CSF here to enable high resolution and mass accuracy of MS detection and facilitate deep structural insights of peptides.

The application of mathematics to biology is not a novelty and just like the discovery of the microscope created a revolution in biology by revealing otherwise invisible and previously unknown worlds, mathematics could also be regarded as a form of microscope which can be used to visualise highly complex data. Today, when Mass-Spectrometry based proteomic assays are becoming standard to identify microorganisms at the genus and species level in many clinical microbiology laboratories, it is prime time to find solutions which would solve the classification problem. In our lab, we are developing one such solution by replacing numerous unrelated single protein searches with something we call a proteom fingerprint. By applying novel mathematics models we can display invisible worlds hidden within forests of data generated by mass spectrometry based proteomics. By diving each protein into n-grams and making a vector of n-gram frequencies we can represent species proteome using term vector model. CUR matrix decomposition, used as a tool for exploratory data analysis, suggests that there are less then 1% of particulary important or influential n-grams in low-dimensional classification problem. Therefore, we propose a proficient dimensionality reduction-based algorithm to identify microorganisms on both species and strain level. Singular Value Decomposition (SVD) often used by Latent Semantic Indexing (LSI) is used to obtain a reduced data representation, therefore solving the sparsity and scalability issues. As a result, significant improvements in computational time are observed together with increased quality of returned results and decreased memory requirements. To show the efficiency of our algorithm, we employed it to bacterial proteomes downloaded from NCBI. The algorithm assigned correctly both strain and species in more than 95% of tested samples. This work was funded by HRZZ (Croatian Science Foundation) research project Clinical proteomics of microorganisms.

ProteinReader (PT) is an expert software developed for the analysis of spectra produced with patented in-solution CAF-/CAF+ peptide derivatization reagent. PT is developed as client-server application to analyse MS/MS spectra on MALDI TOF/TOF mass spec. Four main advantages of CAF-/CAF+ technology are: Allows to capture same peptide in positive and negative mode of operation additional accuracy control In positive mode of operation obtaining mostly Y-ions while in negative mode obtaining mostly B-ions - eased de novo readout No degradation of the sample due to use of CAF-/CAF+ derivatisation reagent Except unambiguous biotypization (species identification), antibiotic resistance can be easily find by aforementioned method ProteinReader uses two methods for peptide search: database match (NCBInr) and de novo reading. Each peptide can be read with the two spectra, in the positive and the negative ion mode. Testing of more than 200 microorganisms showed positive identification on the species level (strain was also successfully identified if present in NCBInr database). ProteinReader was developed using open source technologies (Java, Eclipse RCP, Linux, Apache Tomcat, Infobright data warehouse) and is designed to work effectively (parallel processing) in a multitier client-server architecture. CAF-/CAF+ patent: http://www.google.com/patents/EP2529233A2?cl=en PBF Section for Bioinformatics: http://proteinreader.com/ IRB Laboratory for system biomedicine: http://www.irb.hr/eng/Research/Divisions-and-Centers/Division-of-Molecular-Medicine/Laboratory-for-system-biomedicine This work was funded by HRZZ (Croatian Science Foundation) research project Clinical proteomics of microorganisms.

Peptide mass fingerprinting is a term which describes technique which utilizes ESI or MALDI MS followed by tandem mass spectrometry sequencing. This technique has become a cornerstone for protein identification. Today, applications using peptide mass fingerprinting in biomedical analyses are a major driving force behind its rapid development. However, efficient and accurate analyses of generally big protein tandem mass spectrometry data sets require robust software. In terms of final goal, which is data interpretation, the role of software and underlying algortihms is at least equally important as the technique itself, a fact which is often neglected. High-throughput mass spectrometry instruments can readily generate hundreds of thousands of spectra. This fact combined with the ever growing size of genomic databases imposes tremendous demands for potential successful softvare solutions. In fact, it is the process of comparing large-scale mass spectrometry data with large databases that remains the toughest bottleneck in proteomics. Here we present a completely novel approach based on natural language processing which is not just another improvement of existing approaches, but represents a paradigm shift. It doesn't rely on peak intesity for database peptide matching and it uses newly developed concept of microbial proteome fingerptints for strain/species identification. Since this new algorithm doesn't rely on sequence alignment but instead utilizes a concept of singular proteome fingerprints rather than sets of unrelated peptides, it proposes an elegant solution for this most troubling step in proteome analyses. Abandoning BLAST and other alignment based methods. results in far superior processing speed, accuracy and sensitivity. The above mentioned algortithm can be used to analyse not only proteomes but also metaproteomes coming from mixed microbe communities as in the case presented human urine samples taken from a hospital. The method itself is completely generic, not developed with any specific platform in mind, which makes it highly versatile, able to turn any existing device into highly efficient metaproteome analyzer without siginificant costs related to purchase of new equipment. This work was funded by HRZZ (Croatian Science Foundation) research project Clinical proteomics of microorganisms.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.

The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4, 5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C-18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4, 5-dihydro-1H- imidazole. Complete tagging of the peptides by on-column reaction could be obtained when using D2O, as compared to water-based reaction. Methylation of cysteine residues was enhanced when beta-mercaptoethanol was added in the reactant solution.

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the on-line separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasacharide differing from the acarbose in a number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the off-line one- and two-dimensional NMR experiments and atom assignment was made.

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estrogen.

Pregledom su objašnjeni način rada i principi razdvajanja čestica tehnikom razdvajanja protočnim poljem. Opisana je podjela ove tehnike s obzirom na svojstva analiziranih čestica i primijenjeno vanjsko polje na sedimentacijsku, protočnu, termičku, električnu i magnetsku. Pri tome su za svaku od navedenih tehnika razdvajanja protočnim poljem navedene prednosti, nedostatci i područja primjene. U ovom je radu naveden niz primjera uspješne primjene opisane tehnike. Također su navedeni načini sprezanja tehnika razdvajanja protočnim poljem s jednostavnijim i sa složenijim sustavima detekcije, kao što su detekcija ultraljubičasto-vidljivim spektrom, detekcija višekutnim svjetlosnim raspršenjem ili tehnike spektrometrije masa, pri čemu je na primjerima objašnjena nji- hova uspješna primjena u analizi kompleksnih bioloških uzoraka. Ključne

Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of cereulide in rice and pasta dishes. However, the impact of an exposure to low doses of cereulide was never studied before. In this research, we investigate the effect of low concentrations of cereulide on the behavior of intestinal cells using Caco-2 cell line. The MTT (mitochondrial 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) and the SRB (Sulforhodamine B) reactions were used to measure the mitochondrial activity and the measurement of cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in MTT reaction 1 ng/mL after 3 days of treatment ; in SRB reaction 0.125 ng/mL after 3 days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of cereulide. Additionally, 0.5 and 2 ng/mL of CER increased lactate presence in the cell culture medium. Proteomic data showed that cereulide at concentration of 1 ng/mL led towards a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst first reports to describe the influence of sub-emetic concentrations of cereulide on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

In this study cellular proteins of in vitro grown horseradish (Armoracia lapathifolia Gilib.) tissues of different morphologies, leaves (L), teratoma (TM) and tumor (TN), were separated by one-dimensional (1-DE) and two-dimensional polyacrylamide gel electrophoresis (2-DE) in order to reveal developmentally specific proteins. Moreover, proteins were subjected to analyses by lectin assays to reveal whether the morphological differences were reflected in protein glycosylation. 1-DE glycoprotein pattern showed substantial distinctions between horseradish tissues of different morphologies, which was in contrast to silver stained protein profiles, where only slightly qualitative changes were observed. 2-DE revealed more differences in expression of cellular proteins between examined tissues in comparison to 1-DE, while 2-DE blots confirmed quantitative difference in expression of 43 kDa glycoprotein between investigated tissues. Mass spectrometry analysis identified this protein as a typical horseradish peroxidase. Obtained results imply that the oligosaccharide structures could be important determinants of specificities of horseradish tissue morphology. (doi: 10.5562/cca2078)

The paper presents a detailed study of the biological effects of two amino acid hydroxyurea derivatives – that showed selective antiproliferative effects in vitro on the growth of human tumor cell line SW620. Tested compounds induced cell cycle perturbations and apoptosis. Proteins were identified by proteomics analyses using two-dimensional gel electrophoresis coupled to mass spectrometry, which provided a complete insight into the most probable mechanism of action on the protein level. Molecular targets for tested compounds were analyzed by cheminformatics tools. Zinc-dependent histone deacetylases were identified as potential targets responsible for the observed antiproliferative effect.

The aim of this study is to evaluate the size and stability of radiolytically produced silver clusters. The clusters obtained by the gamma irradiation of a mixture of a silver salt and polyvinyl alcohol (PVA) were studied both theoretically and by mass spectrometry. MALDI TOF mass spectrometry of the Ag-n clusters showed the presence of clusters containing between 5 and 29 atoms. Both the theoretical and experimental results of this paper show that the most stable clusters are small clusters with an odd number of atoms. The stability of these clusters is correlated with the clusters electronic configuration. (C) 2012 Elsevier Ltd. All rights reserved.

Amino acid sequencing and more detailed structure elucidation analysis of peptides and small proteins is a very difficult task even if state-of-the-art mass spectrometry (MS) is employed. To make this task easier, chemical derivatization methods of the N terminus with 4-sulfophenyl-isothiocyanate (SPITC) or the C terminus with 2-methoxy-4,5-dihydro-1H-imidazole (Lys-tag) can enhance peptide fragmentation or fragment ionizability, via proton mobility/localization mechanisms making tandem MS (MS(2)) spectra more informative and less demanding for structural interpretation. Observed disadvantages related to both derivatization methods are sample- and time-consuming procedures and the increased number of reaction byproducts. A novel, sulfate radical in-source formation method of matrix-assisted laser desorption ionization (MALDI) MS based on chemically enhanced in-source decay (ISD) can be accomplished by simple addition of ammonium persulfate (APS) in the matrix solution. This method enables effective decomposition of peptide ions already in the first stage of MS analysis where a large number of fragment ions are produced. The resultant MALDI-ISD mass spectra (MS after APS → MALDI-ISD MS) are almost equivalent to conventional, collision-induced dissociation (CID) MS(2) spectra. These fragment ions are further subjected to the second stage of the MS, and consequently, MS(3) spectra are produced, which makes the sequence analysis more informative and complete (CID MS(2) is thus equivalent to CID MS(3)). Multiply stage MS after APS addition showed enhanced sensitivity, resolution, and mass accuracy compared to peptide derivatization (SPITC and Lys-tag) or conventional MS and MS(2) analyses and offered more detailed insight into peptide structure.

We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation.The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation.The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells.Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article.

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stressresistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non- toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.

The α(v) -integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie and adenovirus receptor. However, until now, it has been unclear whether disulfide bonds in the RGD4C motif influence the retargeting potential of RGD4C-modified Ad5.Replication deficient Ad5 bearing wild-type fiber (Ad5wt) or RGD4G, RGD4C and RGD2C2G insertions within the HI loop of the fiber protein (Ad5RGD4G, Ad5RGD4C and Ad5RGD2C2G, respectively) were used to transduce a panel of cancer cell lines, with or without previous treatment of these Ad5s with the reducing agent dithiothreitol (DTT). In parallel, native and DTT-treated fiber proteins isolated from purified Ad5RGD4C were compared by mass spectrometry.Ad5RGD4C transduced all studied cell lines much more efficiently than Ad5wt, whereas Ad5RGD4G transduced cells only slightly more efficiently than Ad5wt. DTT treatment had no effect on cell transduction by wild-type Ad5wt and Ad5RGD4G but abolished the increased transduction efficacy of Ad5RGD4C in a dose-dependent manner. The mass spectra of native and DTT-reduced tryptic digests of the Ad5RGD4C fiber protein are consistent with the presence of a C(547) -C(549) linkage in the C(547) DC(549) RGDC(553) FC(555) motif. Finally, the high transduction efficacy of Ad5RGD4C is conserved in Ad5RGD2C2G.We provide genetic and biochemical data strongly suggesting that cysteines C(547) and C(549) from the C(547) DC(549) RGDC(553) FC(555) motif inserted in the HI loop of the Ad5 fiber form a single disulfide bond, with this disulfide bond being crucial for Ad5RGD4C retargeting to av-integrins.