A new carbon-based magnetic material for the dispersive solid-phase extraction of UV filters from water samples before liquid chromatography-tandem mass spectrometry analysis.

Magnetic solid-phase extraction is one of the most promising new extraction methods for liquid samples before ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Several types of materials, including carbonaceous ones, have been prepared for this purpose. In this paper, for the first time, the preparation, characterization, and sorption capability of Fe3O4-graphitized carbon black (mGCB) composite toward some compounds of environmental interest were investigated. The synthesized mGCB consisted of micrometric GCB particles with 55 m2 g-1 surface area bearing some carbonyl and hydroxyl functionalities and the surface partially decorated by Fe3O4 microparticles. The prepared mGCB was firstly tested as an adsorbent for the extraction from surface water of 50 pollutants, including estrogens, perfluoroalkyl compounds, UV filters, and quinolones. The material showed good affinity to many of the tested compounds, except carboxylates and glucoronates; however, some compounds were difficult to desorb. Ten UV filters belonging to the chemical classes of benzophenones and p-aminobenzoates were selected, and parameters were optimized for the extraction of these compounds from surface water before UHPLC-MS/MS determination. Then, the method was validated in terms of linearity, trueness, intra-laboratory precision, and detection and quantification limits. In summary, the method performance (trueness, expressed as analytical recovery, 85-114%; RSD 5-15%) appears suitable for the determination of the selected compounds at the level of 10-100 ng L-1, with detection limits in the range of 1-5 ng L-1. Finally, the new method was compared with a published one, based on conventional solid-phase extraction with GCB, showing similar performance in real sample analysis. Graphical Abstract Workflow of the analytical method based on magnetic solid-phase extraction followed by LC-MS/MS determination.

Comprehensive polyphenol profiling of a strawberry extract (Fragaria × ananassa) by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

The aim of metabolic untargeted profiling is to detect and identify unknown compounds in a biological matrix to achieve the most comprehensive metabolic coverage. In phytochemical mixtures, however, the complexity of the sample could present significant difficulties in compound identification. In this case, the optimization of both the chromatographic and the mass-spectrometric conditions is supposed to be crucial for the detection and identification of the largest number of compounds. In this work, a systematic investigation of different chromatographic and mass-spectrometric conditions is presented to achieve a comprehensive untargeted profiling of a strawberry extract (Fragaria × ananassa). To fulfill this aim, an ultra-high-pressure liquid chromatography system coupled via an electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer was used. Spectra were acquired in data-dependent mode, and several parameters were investigated to acquire the largest possible number of both mass spectrometry (MS) features and MS2 mass spectra for unique metabolites. The main classes of polyphenols studied were flavonoids, phenolic acids, dihydrochalcones, ellagitannins, and proanthocyanidins. Method optimization allowed to us identify and tentatively identify 18 and 113 compounds, respectively, among which 74 have never been reported before in strawberries and, to the best of our knowledge, 22 of them have never been reported before. The results show the importance of an extended investigation of the chromatographic and mass-spectrometric method before a complete untargeted profiling of complex phytochemical mixtures.

Recent trends in the analysis of bioactive peptides in milk and dairy products.

Food-derived constituents represent important sources of several classes of bioactive compounds. Among them peptides have gained great attention in the last two decades thanks to the scientific evidence of their beneficial effects on health in addition to their established nutritional value. Several functionalities for bioactive peptides have been described, including antioxidative, antihypertensive, anti-inflammatory, immunomodulatory, and antimicrobial activity. They are now considered as novel and potential dietary ingredients to promote human health, though in some cases they may also have detrimental effects on health. Bioactive peptides can be naturally occurring, produced in vitro by enzymatic hydrolysis, and formed in vivo during gastrointestinal digestion of proteins. Thus, the need to gain a better understanding of the positive health effects of food peptides has prompted the development of analytical strategies for their isolation, separation, and identification in complex food matrices. Dairy products and milk are potential sources of bioactive peptides: several of them possess extra-nutritional physiological functions that qualify them to be classified under the functional food label. In this trends article we briefly describe the state-of-the-art of peptidomics methods for the identification and discovery of bioactive peptides, also considering recent progress in their analysis and highlighting the difficulty in the analysis of short amino acid sequences and endogenous peptides.

Polydopamine-coated magnetic nanoparticles for isolation and enrichment of estrogenic compounds from surface water samples followed by liquid chromatography-tandem mass spectrometry determination.

Estrogens, phytoestrogens, and mycoestrogens may enter into the surface waters from different sources, such as effluents of municipal wastewater treatment plants, industrial plants, and animal farms and runoff from agricultural areas. In this work, a multiresidue analytical method for the determination of 17 natural estrogenic compounds, including four steroid estrogens, six mycoestrogens, and seven phytoestrogens, in river water samples has been developed. (Fe3O4)-based magnetic nanoparticles coated by polydopamine (Fe3O4@pDA) were used for dispersive solid-phase extraction, and the final extract was analyzed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The Fe3O4 magnetic nanoparticles were prepared by a co-precipitation procedure, coated by pDA, and characterized by scanning electron microscopy, infrared spectroscopy, and elemental analysis. The sample preparation method was optimized in terms of extraction recovery, matrix effect, selectivity, trueness, precision, method limits of detection, and method limits of quantification (MLOQs). For all the 17 analytes, recoveries were >70 % and matrix effects were below 30 % when 25 mL of river water sample was treated with 90 mg of Fe3O4@pDA nanoparticles. Selectivity was tested by spiking river water samples with 50 other compounds (mycotoxins, antibacterials, conjugated hormones, UV filters, alkylphenols, etc.), and only aflatoxins and some benzophenones showed recoveries >60 %. This method proved to be simple and robust and allowed the determination of natural estrogenic compounds belonging to different classes in surface waters with MLOQs ranging between 0.003 and 0.1 µg L(-1). Graphical Abstract Determination of natural estrogenic compounds in water by magnetic solid phase extraction followed by liquid chromatography-tandem mass spectrometry analysis.

Purification and identification of endogenous antioxidant and ACE-inhibitory peptides from donkey milk by multidimensional liquid chromatography and nanoHPLC-high resolution mass spectrometry.

Donkey milk is a valuable product for the food industry due to its nutraceutical, nutritional, and functional properties. In this work, the endogenous peptides from donkey milk were investigated for their antioxidant and ACE-inhibitory activities, combining a two-dimensional peptide fractionation strategy with high-resolution mass spectrometry, bioinformatics analysis, and in vitro assays. After extraction, the endogenous peptides were fractionated twice, first by polymeric reversed phase and then by hydrophilic interaction chromatography. Fractions were screened for the investigated bioactivities and only the most active ones were finally analyzed by nanoRP-HPLC-MS/MS; this approach allowed to reduce the total number of possible bioactive sequences. Results were further mined by in silico analysis using PeptideRanker, BioPep, and PepBank, which provided a bioactivity score to the identified peptides and matched sequences to known bioactive peptides, in order to select candidates for chemical synthesis. Thus, five peptides were prepared and then compared to the natural occurring ones, checking their retention times and fragmentation patterns in donkey milk alone and in spiked donkey milk samples. Pure peptide standards were finally in vitro tested for the specific bioactivity. In this way, two novel endogenous antioxidant peptides, namely EWFTFLKEAGQGAKDMWR and GQGAKDMWR, and two ACE-inhibitory peptides, namely REWFTFLK and MPFLKSPIVPF, were successfully validated from donkey milk. Graphical Abstract Analytical workflow for purification and identification of bioactive peptides from donkey milk sample.

Mycoestrogen determination in cow milk: Magnetic solid-phase extraction followed by liquid chromatography and tandem mass spectrometry analysis.

Recently, magnetic solid-phase extraction has gained interest because it presents various operational advantages over classical solid-phase extraction. Furthermore, magnetic nanoparticles are easy to prepare, and various materials can be used in their synthesis. In the literature, there are only few studies on the determination of mycoestrogens in milk, although their carryover in milk has occurred. In this work, we wanted to develop the first (to the best of our knowledge) magnetic solid-phase extraction protocol for six mycoestrogens from milk, followed by liquid chromatography and tandem mass spectrometry analysis. Magnetic graphitized carbon black was chosen as the adsorbent, as this carbonaceous material, which is very different from the most diffuse graphene and carbon nanotubes, had already shown selectivity towards estrogenic compounds in milk. The graphitized carbon black was decorated with Fe3 O4 , which was confirmed by the characterization analyses. A milk deproteinization step was avoided, using only a suitable dilution in phosphate buffer as sample pretreatment. The overall process efficiency ranged between 52 and 102%, whereas the matrix effect considered as signal suppression was below 33% for all the analytes even at the lowest spiking level. The obtained method limits of quantification were below those of other published methods that employ classical solid-phase extraction protocols.

Ultra-high-performance liquid chromatography-tandem mass spectrometry for the analysis of free and conjugated natural estrogens in cow milk without deconjugation.

A sensitive liquid chromatography/electrospray ionization-tandem mass spectrometry method for the determination of free and conjugated estrogens in cow milk is described. Milk samples were diluted with water and then extracted and cleaned up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization, and/or protein precipitation step. Two fractions were collected (free and conjugated estrogens) and analyzed separately. Mass spectrometry analysis was performed in negative ion mode using selected reaction monitoring acquisition mode. For all compounds, the coefficients of determination (R(2)) ranged between 0.9892 and 0.9997. Analytical recoveries were in the range of 86-109% for free estrogens and 85-118% for conjugates, with relative standard deviations below 10%, and the method detection limit ranged between 3 and 80 ng L(-1). Finally, the developed method was used to determine the presence of free and conjugated estrogens in six retail milk samples (five cow milk samples and one goat milk sample) and one goat milk sample provided by a local shepherd. Estrone was found to be the major free estrogen present in commercial milk samples, and estrone 3-sulfate showed the highest concentration among the target conjugated estrogens. Estriol was also observed in some analyzed milk samples, but the concentrations were always below the limit of quantification.

Development of an analytical strategy for the identification of potential bioactive peptides generated by in vitro tryptic digestion of fish muscle proteins.

In the last years, food proteins and peptides are attracting great attention because of the emergence of a new field, that of food-derived bioactive peptides. This paper presents a comparison and evaluation of four different experiments for the identification of sarcoplasmic and myofibrillar fish peptides. This study is aimed at the development of a simple and fast method for the identification of peptides that could arise from fish meat if trypsin was the only digestive enzyme acting on fish meat proteins. In particular, we tested the use of ultrafiltration membranes with a molecular weight cutoff of 3,000 Da. Data analysis has shown that the experiment in which there is neither precipitation nor an ultrafiltration step performed better and allowed the identification of a larger number of peptides and potential antimicrobial peptides (AMPs); this workflow provided 473 and 398 total identified peptides and 44 and 18 AMPs for sarcoplasmic and myofibrillar extracts, respectively. This protocol is found to be faster and more straightforward than the other three tested workflows. The developed strategy could be also useful for other food matrices and could provide information about food quality and safety control.

Natural estrogens in dairy products: Determination of free and conjugated forms by ultra high performance liquid chromatography with tandem mass spectrometry.

Natural estrogens are synthesized by mammals in different amounts depending on the developmental stage and pregnancy/lactation period, and they may pass into milk, where they are mostly present as glucuronated and sulfated forms. In modern dairy practices, about 75% of milk is produced from pregnant cows; therefore, the amount of hormones that may pass into milk could be of concern. While estrogen determination in milk has been investigated in depth, the individual determination of estrogens and their conjugated forms in dairy products has not been fully addressed. The aim of this work was to develop and assess a sensitive method, using the peculiar retention properties of graphitized carbon black, to extract natural free estrogens and their major conjugated metabolites, without any enzymatic cleavage, from yogurt, cheese, and butter. The free and conjugated estrogens were eluted in two distinct fractions from the solid-phase extraction cartridge and analyzed separately by ultra high performance liquid chromatography coupled to tandem mass spectrometry. Recoveries were higher than 80% for all the three sample typologies. The highest matrix effects were observed for butter, which was richest in lipid content, but was below 30%. A survey on some commercial dairy products suggests that production processes decreased estrogen content.

Proteomic study of a tolerant genotype of durum wheat under salt-stress conditions.

Salinity is one of the major abiotic stress conditions limiting crop growth and productivity. Duilio is a wheat genotype that shows tolerant behavior in both salt-stress and drought-stress conditions. Toward better understanding of the biochemical response to salinity in this genotype of durum wheat, a comparative label-free shotgun proteomic analysis based on normalized spectral abundance factors was conducted on wheat leaf samples subjected to increasing salt-stress levels (100 and 200 mmol L(-1) NaCl) with respect to untreated samples. We found significant changes in 71 proteins for the first stress level, in 83 proteins at the higher salinity level, and in 88 proteins when comparing salt-stress levels with each other. The major changes concerned the proteins involved in primary metabolism and production of energy, followed by those involved in protein metabolism and cellular defense mechanisms. Some indications of different specific physiological and defense mechanisms implicated in increasing tolerance were obtained. The enhanced salinity tolerance in Duilio appeared to be governed by a higher capacity for osmotic homeostasis, a more efficient defense, and an improvement of protection from mechanical stress by increased cell wall lignifications, allowing a better potential for growth recovery.

Multiresidue determination of UV filters in water samples by solid-phase extraction and liquid chromatography with tandem mass spectrometry analysis.

UV filters, contained in sunscreens and other cosmetic products, as well as in some plastics and industrial products, are nowadays considered contaminants of emerging concern because their widespread and increasing use has lead to their presence in the environment. Furthermore, some UV filters are suspected to have endocrine disruption activity. In the present work, we developed an analytical method based on liquid chromatography with tandem mass spectrometry for the determination of UV filters in tap and lake waters. Sixteen UV filters were extracted from water samples by solid-phase extraction employing graphitized carbon black as adsorbent material. Handling 200 mL of water sample, satisfactory recoveries were obtained for almost all the analytes. The limits of detection and quantification of the method were comparable to those reported in other works, and ranged between 0.7-3.5 and 1.9-11.8 ng/L, respectively; however in our case the number of investigated compounds was larger. The major encountered problem in method development was to identify the background contamination sources and reduce their contribution. UV filters were not detected in tap water samples, whereas the analyses conducted on samples collected from three different lakes showed that the swimming areas are most subject to UV filter contamination.

Multiclass mycotoxin analysis in food, environmental and biological matrices with chromatography/mass spectrometry.

Mold metabolites that can elicit deleterious effects on other organisms are classified as mycotoxins. Human exposure to mycotoxins occurs mostly through the intake of contaminated agricultural products or residues due to carry over or metabolite products in foods of animal origin such as milk and eggs, but can also occur by dermal contact and inhalation. Mycotoxins contained in moldy foods, but also in damp interiors, can cause diseases in humans and animals. Nephropathy, various types of cancer, alimentary toxic aleukia, hepatic diseases, various hemorrhagic syndromes, and immune and neurological disorders are the most common diseases that can be related to mycotoxicosis. The absence or presence of mold infestation and its propagation are seldom correlated with mycotoxin presence. Mycotoxins must be determined directly, and suitable analytical methods are necessary. Hundreds of mycotoxins have been recognized, but only for a few of them, and in a restricted number of utilities, a maximum acceptable level has been regulated by law. However, mycotoxins seldom develop alone; more often various types and/or classes form in the same substrate. The co-occurrence might render the individual mycotoxin tolerance dose irrelevant, and therefore the mere presence of multiple mycotoxins should be considered a risk factor. The advantage of chromatography/mass spectrometry (MS) is that many compounds can be determined and confirmed in one analysis. This review illustrates the state-of-the-art of mycotoxin MS-based analytical methods for multiclass, multianalyte determination in all the matrices in which they appear. A chapter is devoted to the history of the long-standing coexistence and interaction among humans, domestic animals and mycotoxicosis, and the history of the discovery of mycotoxins. Quality assurance, although this topic relates to analytical chemistry in general, has been also examined for mycotoxin analysis as a preliminary to the systematic literature excursus. Sample handling is a crucial step to devise a multiclass analytical method; so when possible, it has been treated separately for a better comparison before tackling the instrumental part of the whole analytical method. This structure has resulted sometimes in unavoidable redundancies, because it was also important to underline the interconnection. Most reviews do not deal with all the possible mycotoxin sources, including the environmental ones. The focus of this review is the analytical methods based on MS for multimycotoxin class determination. Because the final purpose to devise multimycotoxin analysis should be the assessment of the danger to health of exposition to multitoxicants of natural origin (and possibly also the interaction with anthropogenic contaminants), therefore also the analytical methods for environmental relevant mycotoxins have been thoroughly reviewed. Finally, because the best way to shed light on actual risk assessment could be the individuation of exposure biomarkers, the review covers also the scarce literature on biological fluids.

Determination of enantioselectivity and enantiomeric excess by mass spectrometry in the absence of chiral chromatographic separation: an overview.

This review describes the principles and instrumentation for the screening of asymmetric reactions by mass spectrometry. These techniques witnessed a significant advancement in the last few years. Although some of them are still at the proof-of-concept development stage, several applications might be foreseen in the field of combinatorial, high-throughput parallel catalyst screening.

Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins.

A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA-cationic lipid complexes (lipoplexes), and lipid-polycation-DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.

Proteome investigation of the non-model plant pomegranate (Punica granatum L.).

A gel-free, shotgun proteomics approach was used to characterize pomegranate aril proteome by nanoliquid chromatography-high-resolution tandem mass spectrometry. To identify both high-abundance and low-abundance proteins, we applied two distinct sample preparation protocols, i.e., a classical one widely applied in literature and a second one able to reduce the dynamic range of protein concentration of the sample, based on combinatorial hexapeptide ligand library technology. However, the proteins identified with the latter protocol were only a small minority. Because pomegranate is a non-model plant species, i.e., information of its genome sequence are lacking, only a few protein sequences are included in the most widely known protein sequence databases. To improve both the number of identified proteins and data reliability, identification was performed integrating the results obtained with three distinct plant protein databases, since the majority of proteins could only be attributed by homology with other plant species. Nevertheless, many proteins had assigned only one unique peptide, because of the phylogenetic distance of pomegranate from the main model plants. After manual revision of the identified proteins to eliminate the redundant or ambiguous identifications, a list of 1,488 proteins was obtained, only six of which belonging to pomegranate species. To the author's best knowledge, this is the first work aimed at the proteomic characterization of Punica granatum.

DNA affects the composition of lipoplex protein corona: a proteomics approach.

The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.

Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems.

A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.

Evaluation of different two-dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue.

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large-scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide-level fractionation, the first dimension of separation was performed with high-pH reversed-phase chromatography (pH-RP) and strong cation exchange chromatography (SCX). Regarding protein-level fractionation, C(8) protein reversed-phase (C(8) -RP Prot) and high-recovery protein reversed-phase (hr-RP Prot) were used instead. The second dimension consisted of a reversed-phase nano-HPLC on-Chip coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide-level pH-RP and the hr-RP Prot protein-level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide-level SCX, with 509 proteins identified, was the worst method.

Immunoprecipitation on magnetic beads and liquid chromatography-tandem mass spectrometry for carbonic anhydrase II quantification in human serum.

In this study, a magnetic bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) operating in multiple reaction monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis, and calibration, were optimized. The method was validated in terms of recovery (77%), reproducibility (relative standard deviation [RSD]<12%), and method detection limit (0.5 pmol ml(-1)). The developed method was applied to determining the CA II in eight healthy subjects, and the concentration measured was 27.3 pmol ml(-1) (RSD = 65%).

Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer.

Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the "protein corona" absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle-protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids.