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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.

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.

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.

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.

Due to the wide and increasing application of engineered silver nanoparticles (AgNPs) in commercial products, their release and disposal into aquatic environmental compartments is unavoidable. The final environmental fate of AgNPs depends on their stability, behavior and lifetime in a particular system. This study aimed to systematically investigate the aggregation and dissolution behavior of AgNPs in model aquatic systems including a range of different pH values, different concentrations of mono- and divalent electrolytes, and the presence of non-ionic, anionic and cationic surfactants used in commercial detergents. The investigation was performed by a combination of surface plasmon resonance (SPR) techniques, dynamic light scattering (DLS) and electrophoretic light scattering (ELS) methods, inductively coupled plasma mass spectroscopy (ICPMS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The obtained results highlighted the importance of pH, ionic strength and interaction with surfactants relevant to the laundry cycle to the stability and environmental fate of citrate-coated AgNPs. An important implication of this study is that the interaction of AgNPs with surfactants present in wastewater effluents could potentially become more environmentally hazardous due to the increased persistence and dissolution of AgNPs.

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.

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a pH around 11. This method enables separation and accurate detection of adenylates. Our study provides new insight into the complexity of adenylate extraction and quantification that is crucial for the application of adenylate energy charge as a confident physiological measure of environmental stress and as a health index of G. fossarum.

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.

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]

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.

Using high resolution molecular technique of atomic force microscopy, we address the extracellular polymer production of Adriatic diatom Cylindrotheca closterium analyzed at the single cell level and the supramolecular organization of gel phase isolated from the Northern Adriatic macroaggregates. Our results revealed that extracellular polysaccharides freshly produced by marine diatoms can self-assemble directly to form gel network characteristics of the macroscopic gel phase in the natural aquatorium. Based on the experiments performed with isolated polysaccharide fractions of C. closterium and of macroaggregates gel phase, we demonstrated that the polysaccharide self-assembly into gel network can proceed independent of any bacterial mediation or interaction with inorganic particles.

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.

In conclusion, the significance of single adhesion events at the mercury electrode has been apparent for more than two decades, since the discovery of stochastic adhesion signals of vesicles in seawater samples.39-41 An impressive subsequent progress made in the time-resolved data acquisition of stochastic amperometric signals has been illustrated by the H & S experiments on liposome adhesion at the static mercury drop electrode.1 However, this achievement is only a prerequisite for a consistent analysis and interpretation of the adhesion signals of liposomes in order to extract the kinetic data that are not accessible by other methods.

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.

Extracellular polysaccharide production by marine diatoms is a significant route by which photosynthetically produced organic carbon enters the trophic web and may influence the physical environment in the sea. This study highlights the capacity of atomic force microscopy (AFM) for investigating diatom extracellular polysaccharides with a subnanometer resolution. Here we address a ubiquitous marine diatom Cylindrotheca closterium, isolated from the northern Adriatic Sea, and its extracellular polymeric substance (EPS) at a single cell level. We applied a simple procedure for AFM imaging of diatom cells on mica under ambient conditions (in air) to achieve visualization of their EPS with molecular resolution. The EPS represents a web of polysaccharide fibrils with two types of cross-linking: fibrils association forming junction zones and fibril–globule interconnections with globules connecting two or more fibrils. The fibril heights were 0.4–2.6 nm while globules height was in the range of 3–12 nm. Polymer networks of native gel samples from the Northern Adriatic and the network formed by polysaccharides extracted from the C. closterium culture share the same features regarding the fibril heights, pore openings and the mode of fibril association, proving that the macroscopic gel phase in the Northern Adriatic can be formed directly by the self-assembly of diatom released polysaccharide fibrils. Copyright © 2011 John Wiley & Sons, Ltd.

We present the first atomic force microscopy (AFM) images of the native marine gel network and a new type of electrochemical signals of individual gel microparticles in seawater. Gel microparticles in seawater are selectively detected through specific amperometric signals using a dropping mercury electrode (DME) as a sensor. We have demonstrated that organic microparticles naturally present in Northern Adriatic seawater can be detected as single particles and sorted at the DME/seawater interface according to their hydrophobicity and supramolecular organization., Prvi puta prikazana je nanostruktura morskog gela oslikana mikroskopijom atomskih sila i nova vrsta elektrokemijskih signala mikročestica gela u morskoj vodi. Čestice gela u morskoj vodi selektivno se detektiraju kao specifični amperometrijski signali primjenom živine kapajuće elektrode kao senzora. Svaki signal je rezultat sudara mikročestice gela s rastućom površinom živine elektrode. Primjenom amperometrijske tehnike i živine kapajuće elektrode kao senzora organske mikročestice detektiraju se na dinamičkoj međupovršini živina elektroda/morska voda temeljem svojstava hidrofobnosti i supramolekulske organizacije kao površinski-aktivne i gel čestice.

The macroscopic gel phase (mucilage) appears episodically in the northern Adriatic Sea covering tens of square kilometers of sea surface. Current views leave no doubt on phytoplankton production as a proximal source of polymers constituting the gel network, but the mechanism leading to its rapid production remains unknown. We introduced electrochemical sensing of marine microparticles and atomic force microscopy to image supramolecular organization of marine gel network. Our biophysical scenario of mucilage event features self-organization of biopolymers into microparticles ("marine vesicles") that under specific conditions transform to giant gel by a fast vesicle-to-gel transition.

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 microC/cm2 in 0.1 M NaCl and 1 M NaCl solutions, respectively.