First Proteome Analysis of Poplar-Type Propolis.

Propolis is a natural mixture of honeybee-released and plant-derived compounds produced by honeybees. Poplar propolis is rich in bioactive polyphenolic compounds, and due to its many health benefits, it is commonly used as a food supplement or functional food ingredient. However, it is the only honeybee product whose proteome hasn't been analyzed. Here, we report a first proteome analysis of poplar-type propolis, a challenging glue-type resinous sample for protein characterization. Raw propolis mixture was precipitated with cold acetone to obtain the protein fraction. Proteins were digested with trypsin, and generated peptides were analyzed on nano-ESI-qTOF SYNAPT G2-Si mass spectrometer (MS) by data-independent acquisition (DIA) and data-dependent acquisition (DDA). Identified peptides and inferred proteins suggest the presence of new bioactive molecules as components of propolis. The poplar-type propolis proteome is composed of a mixture of proteins from the Apis and Populus genera. This is the first-ever report of the proteome of any type of propolis.

N-Terminal chemical derivatization of peptides with 4-formyl-benzenesulfonic acid and electrospray positive and negative tandem mass spectrometry with high abundance of b-ion series.

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.

Different chemical proteomic approaches to identify the targets of lapatinib.

The process of identifying the protein targets and off-targets of a biologically active compound is of great importance in modern drug discovery. Various chemical proteomics approaches have been established for this purpose. To compare the different approaches, and to understand which method would provide the best results, we have chosen the EGFR inhibitor lapatinib as an example molecule. Lapatinib derivatives were designed using linkers with motifs, including amino (amidation), alkyne (click chemistry) and the diazirine group (photo-affinity). These modified lapatinib analogues were validated for their ability to inhibit EGFR activity in vitro and were shown to pull down purified recombinant EGFR protein. In all of the approaches evaluated here, we identified EGFR as the main protein target from the lysate of immortalised cell line expressing EGFR, thus validating its potential use to identify unknown protein targets. Taken together, the results reported here give insight into the cellular activities of lapatinib.

Integrin αvß3 and disulfide bonds play important roles in NGR-retargeted adenovirus transduction efficiency.

AIMS: Adenoviruses that have CNGRCVSGCAGRC 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 CNGRCVSGCAGRC sequence inserted into Ad serotype 5 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 αvß3 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 αvß3 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 αvß3 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.

NanoUPLC-QTOF-MS/MS Determination of Major Rosuvastatin Degradation Products Generated by Gamma Radiation in Aqueous Solution.

Rosuvastatin, a member of the statin family of drugs, is used to regulate high cholesterol levels in the human body. Moreover, rosuvastatin and other statins demonstrate a protective role against free radical-induced oxidative stress. Our research aimed to investigate the end-products of free radical-induced degradation of rosuvastatin. To induce the radical degradation, an aqueous solution of rosuvastatin was irradiated using different doses of gamma radiation (50-1000 Gy) under oxidative conditions. Rosuvastatin and related degradation products were separated on nanoC18 column under gradient elution, and identification was carried out on hyphenated nanoUPLC and nanoESI-QTOF mass spectrometer system. Elemental composition analysis using highly accurate mass measurements together with isotope fitting algorithm identified nine major degradation products. This is the first study of gamma radiation-induced degradation of rosuvastatin, where chemical structures, MS/MS fragmentation pathways and formation mechanisms of the resulting degradation products are detailly described. The presented results contribute to the understanding of the degradation pathway of rosuvastatin and possibly other statins under gamma radiation conditions.

Study of the venom proteome of Vipera ammodytes ammodytes (Linnaeus, 1758): A qualitative overview, biochemical and biological profiling.

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.

Identification of pathogens from native urine samples by MALDI-TOF/TOF tandem mass spectrometry.

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.

Analysis of Fatty Acid Esters of Hydroxyl Fatty Acid in Selected Plant Food.

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.

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.

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.

High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions.

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.

Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin.

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±0.43mol 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.

A novel type of colony formation in marine planktonic diatoms revealed by atomic force microscopy.

Diatoms have evolved a variety of colonial life forms in which cells are connected by organic threads, mucilage pads or silicate structures. In this study, we provide the first description of a novel strategy of colony formation among marine planktonic diatoms. Bacteriastrum jadranum forms loose but regular chains with distinct heterovalvate terminal cells. The colonial cells and their siliceous projections, the setae, are not in direct contact; instead, they are enclosed within the optically transparent organic matrix. This cell jacket structure was detected by staining procedure with Alcian Blue, which showed that the polysaccharides are predominant matrix constituents and revealed that the jacket reaches the span of the setae. The scanning electron microscopy (SEM) observations showed distinguishable fibrillar network firmly associated with cells. Using atomic force microscopy (AFM), we were able to visualise and characterise the cell jacket structure at molecular resolution. At nanoscale resolution, the cell jacket appears as a cross-linked fibrillar network organised into a recognisable structure. The circular patches of self-repeating pattern (hexagonal pores with openings of 8-100 nm) are connected through thicker surrounding fibrils and reinforced by branching fibrils. The pore-forming fibrils within the patches are only 0.6-1.6 nm high, the surrounding fibrils connecting patches are 2.0-2.8 nm high, and the branching fibrils are considerably wider but not higher than 4.0 nm. The discovered polysaccharide fibrillar network is highly organised and delicately structured with a monomolecular fibril height of 0.6 nm. We conclude that the Bacteriastrum polysaccharide jacket represents an essential part of the cell, as the conjunction of the polymer network with the frustule appears to be extremely tight and such specific and unique patterns have never been found in self-assembled polysaccharide gel networks, which are usually encountered in the marine environment.

Nanomechanical characterization of the stiffness of eye lens cells: a pilot study.

PURPOSE: The purpose of this study is to probe the mechanical properties of individual eye lens cells isolated from nucleus and cortex of adult sheep eye lens, and to characterize the effect of cytoskeletal drugs. METHODS: We used atomic force microscopy (AFM), featuring a spherical tip at the end of a soft cantilever, to indent single lens cells, and measure the Young's modulus of isolated nuclear and cortical lens cells. Measurements were performed under basal conditions, and after addition of drugs that disrupt actin filaments and microtubules. RESULTS: We found that single lens cells were able to maintain their shape and mechanical properties after being isolated from the lens tissue. The median Young's modulus value for nuclear lens cells (4.83 kPa) was ~ 20-fold higher than for cortical lens cells (0.22 kPa). Surprisingly, disruption of actin filaments and microtubules did not affect the measured Young's moduli. CONCLUSIONS: We found that single cells from the lens nucleus and cortex can be distinguished unambiguously using the elastic modulus as a criterion. The uncommon maintenance of shape and elastic properties after cell isolation together with the null effect of actin filaments and microtubules targeting drugs suggest that the mechanical stability of fiber cells is provided by cellular elements other than the usual cytoskeletal proteins.

Probing cell surface charge by scanning electrode potential.

A static mercury electrode was used for measurement of double-layer charge displacement signals caused by living plant cells of the unicellular marine alga Dunaliella tertiolecta. By scanning the electrode potential a point is reached where the charge density of a plant cell compensates the electrode charge density. The experimentally determined values of surface charges for unicellular marine alga Dunaliella tertiolecta cells are -0.63 and -0.75 nuC/cm(2) in 0.1 M NaCl and 1 M NaCl solutions, respectively.