First Proof of Concept of a Click Inverse Electron Demand Diels-Alder Reaction for Assigning the Regiochemistry of Carbon-Carbon Double Bonds in Untargeted Lipidomics.

Lipidomics by high-resolution mass spectrometry (HRMS) has become a prominent tool in clinical chemistry due to the proven connections between lipid dysregulation and the insurgence of pathologies. However, it is difficult to achieve structural characterization beyond the fatty acid level by HRMS, especially when it comes to the regiochemistry of carbon-carbon double bonds, which play a major role in determining the properties of cell membranes. Several approaches have been proposed for elucidating the regiochemistry of double bonds, such as derivatization before MS analysis by photochemical reactions, which have shown great potential for their versatility but have the unavoidable drawback of splitting the MS signal. Among other possible approaches for derivatizing electron-rich double bonds, the emerging inverse-electron-demand Diels-Alder (IEDDA) reaction with tetrazines stands out for its unmatchable kinetics and has found several applications in basic biology and protein imaging. In this study, a catalyst-free click IEDDA reaction was employed for the first time to pinpoint carbon-carbon double bonds in free and conjugated fatty acids. Fatty acid and glycerophospholipid regioisomers were analyzed alone and in combination, demonstrating that the IEDDA reaction had click character and allowed the obtention of diagnostic product ions following MS/MS fragmentation as well as the possibility of performing relative quantitation of lipid regioisomers. The IEDDA protocol was later employed in an untargeted lipidomics study on plasma samples of patients suffering from prostate cancer and benign prostatic conditions, confirming the applicability of the proposed reaction to complex matrices of clinical interest.

Preparation of Monolith for Online Extraction and LC-MS Analysis of ß-Estradiol in Serum Via a Simple Multicomponent Reaction.

Multicomponent reactions offer efficient and environmentally friendly strategies for preparing monoliths suitable for applications in analytical chemistry. In the described study, a multicomponent reaction was utilized for the one-pot miniaturized preparation of a poly(propargyl amine) polymer inside commercial silica-lined PEEK tubing. The reaction involved only small amounts of reagents and was characterized by atom economy. The resulting monolithic column was incorporated into an autosampler system for the online extraction and cleanup of ß-estradiol from human serum. Sample pretreatment was simplified to a simple dilution with methanol and centrifugation to remove proteins. The resulting platform included LC-MS analysis in multiple reaction monitoring for quantitative analysis of ß-estradiol. The method was validated in serum, demonstrating practical applicability for the monitoring of fertile women. Recoveries were above 94%, and LOD and LOQ values at 0.008 and 0.18 ng mL-1, respectively. The developed platform proved to be competitive with previous methods for solid-phase microextraction of ß-estradiol in serum, with comparable recovery and sensitivity but with the advantage of nearly complete automation. The environmental impact of the process was evaluated as acceptable due to the miniaturization of the monolith synthesis and the automation of extraction. The drawback associated with the LC-MS technique can be reduced by the inclusion of additional analytes in a single investigation. The work demonstrates that multicomponent reactions are versatile, economical, and possibly a green methodology for producing reversed-phase and mixed-mode sorbents, enabling miniaturization of the entire analytical procedure from the preparation of extraction sorbents to analysis.

Recent trends in glycoproteomics by characterization of intact glycopeptides.

This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC-MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC-MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird's-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic.

Dispersive solid phase extraction using a hydrophilic molecularly imprinted polymer for the selective extraction of patulin in apple juice samples.

A molecularly imprinted polymer with a specific selectivity for patulin was successfully synthesized. The molecularly imprinted material was prepared using the two functional monomers dopamine and melamine and formaldehyde as the cross-linker. The resulting material possessed a large number of hydrophilic groups, such as hydroxyls, imino groups, and ether linkages. For the first time, uric acid was used as a dummy template for its structural similarity to patulin. Comprehensive characterization and detailed studies of the adsorption process were carried out via adsorption isotherms, while the rate-limiting steps were investigated using adsorption kinetics. Separation, determination, and quantification of patulin were achieved by ultra-high performance liquid chromatography coupled with both photodiode array detection and tandem mass spectrometry. The latter was applied to patulin confirmation in the analysis of real samples. The methodology was validated in 20 apple juice samples. The results showed that the developed hydrophilic molecularly imprinted polymer had high selectivity and specific adsorption towards patulin, with mean recoveries ranging between 85 and 90% and a relative standard deviation lower than 15%. The developed molecularly imprinted polymer exhibited good linearity in the range 1-100 ng mL-1 with coefficient of determination (R2) > 0.99. The limit of detection was 0.5 ng mL-1, and the limit of quantification was 1 ng g-1. The developed method showed a good purification capacity for apple juices due to its hydrophilic nature and the polar interactions established with the target analyte.

The Key Role of Metal Adducts in the Differentiation of Phosphopeptide from Sulfopeptide Sequences by High-Resolution Mass Spectrometry.

Site localization of protein sulfation by high-throughput proteomics remains challenging despite the technological improvements. In this study, sequence analysis and site localization of sulfation in tryptic peptides were determined under a conventional nano-liquid chromatography-mass spectrometry configuration. Tryptic sulfopeptide standards were used to study different fragmentation strategies, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD), and electron-transfer/collision-induced dissociation (ETciD), in the positive ionization mode. Sulfopeptides displayed only neutral loss of SO3 under CID, while the sequence could be determined for all other tested fragmentation techniques. Results were compared to the same sequences with phosphotyrosine, indicating important differences, as the sequence and modification localization could be studied by all fragmentation strategies. However, the use of metal adducts, especially potassium, provided valuable information for sulfopeptide localization in ETD and ETD-hybrid strategies by stabilizing the modification and increasing the charge state of sulfopeptides. In these conditions, both the sequence and localization could be obtained. In-source neutral loss of SO3 under EThcD provided diagnostic peaks suitable to distinguish the sulfopeptides from the nearly isobaric phosphopeptides. Further confirmation on the modification type was found in the negative ionization mode, where phosphopeptides always had the typical phosphate product ion corresponding to PO3-.

Novel Aza-Paternò-Büchi Reaction Allows Pinpointing Carbon-Carbon Double Bonds in Unsaturated Lipids by Higher Collisional Dissociation.

The evaluation of double bond positions in fatty acyl chains has always been of great concern given their significance in the chemical and biochemical role of lipids. Despite being the foremost technique for lipidomics, it is difficult in practice to obtain identification beyond the fatty acyl level by the sole high-resolution mass spectrometry. Paternò-Büchi reactions of fatty acids (FAs) with ketones have been successfully proposed for pinpointing double bonds in FAs in combination with the collision-induced fragmentation technique. In the present paper, an aza-Paternò-Büchi (aPB) reaction of lipids with 6-azauracil (6-AU) was proposed for the first time for the determination of carbon-carbon double bonds in fatty acyl chains using higher collisional dissociation in the negative ion mode. The method was optimized using free FA and phospholipid analytical standards and compared to the standard Paternò-Büchi reaction with acetone. The introduction of the 6-AU moiety allowed enhancing the ionization efficiency of the FA precursor and diagnostic product ions, thanks to the presence of ionizable sites on the derivatizing agent. Moreover, the aPB derivatization allowed the obtention of deprotonated ions of phosphatidylcholines, thanks to an intramolecular methyl transfer from the phosphocholine polar heads during ionization. The workflow was finally applied for pinpointing carbon-carbon double bonds in 77 polar lipids from an yeast (Saccharomyces cerevisiae) extract.

Stereoselective Synthesis of Spiro-Decalin Oxindole Derivatives via Sequential Organocatalytic Michael-Domino Michael/Aldol Reaction.

A highly stereoselective procedure for the synthesis of spiro-polycyclic oxindoles bearing five contiguous stereogenic centers including two tetrasubstituted carbons has been developed. Under sequential organocatalysis performed by a pyrrolidine-based organocatalyst and DBU, a highly atom-economical Michael-domino Michael/aldol reaction sequence was optimized, yielding variously functionalized spiro-decalin oxindoles with excellent stereoselectivity (>99:1 dr, up to 92% ee).

Detailed investigation of the composition and transformations of phenolic compounds in fresh and fermented Vaccinium floribundum berry extracts by high-resolution mass spectrometry and bioinformatics.

INTRODUCTION: Blueberries are known for their very high content of biologically active phenolic compounds; nonetheless, differently from the North American and European species of blueberries, Neotropical blueberries have not been extensively studied yet. OBJECTIVES: In the present paper, the phenolic composition of Vaccinium floribundum Kunth, which is endemic to the Andean regions and grows 1,600 to 4,500 meters above sea level, was investigated by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Native and fermented berries were compared in terms of phenolic composition as well as antioxidant activity, total phenolic content, and total anthocyanin content. MATERIALS AND METHODS: V. floribundum native and fermented berries were extracted and analyzed by UHPLC-HRMS. The acquired datasets were processed by Compound Discoverer 3.1 using a dedicated data analysis workflow that was specifically set up for phenolic compound identification. RESULTS: In total, 309 compounds were tentatively identified, including anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. The molecular transformations of phenolic compounds during fermentation were comprehensively investigated for the first time, and by a customized data processing workflow, 13 quinones and quinone methides were tentatively identified in the fermented samples. Compared to other species of the genus Vaccinium, a peculiar phenolic profile is observed, with low abundance of highly methylated compounds. CONCLUSION: Andean berries are a rich source of a wide variety of phenolic compounds. Untargeted MS analyses coupled to a dedicated data processing workflow allowed expanding the current knowledge on these berries, improving our understanding of the fate of phenolic compounds after fermentation.

Investigating the Short Peptidome Profile of Italian Dry-Cured Ham at Different Processing Times by High-Resolution Mass Spectrometry and Chemometrics.

Short peptides have been spiking interest owing to their significant contribution to the taste and functional properties of dry-cured ham. In this study, a suspect screening approach based on high-resolution mass spectrometry was employed for the comprehensive characterization of the short endogenous peptidome in dry-cured ham samples at different processing stages (14, 22, and 34 months). After careful manual spectra interpretation, a chemometric approach based on principal component analysis was employed for highlighting the differences between the three sets of samples. A total of 236 short peptide sequences was tentatively identified, including 173 natural short peptides and 63 sequences containing non-proteinogenic amino acids, the highest number ever reported for endogenous sequences in dry-cured ham. Samples in the latest processing stages presented a generally higher abundance of dipeptides, indicating residual proteolytic activity. Moreover, the several annotated modified short peptides, mainly pyroglutamination and lactoyl conjugation, allowed hypothesizing several reactions occurring over time. For the first time, several lactoyl-dipeptides were tentatively identified in dry-cured ham samples with maximum concentration in the late processing stage samples. The presented results significantly contribute to the understanding of the reaction involving short peptides that affect the sensory and functional properties of dry-cured ham.

Fully Automatized Detection of Phosphocholine-Containing Lipids through an Isotopically Labeled Buffer Modification Workflow.

High-resolution mass spectrometry is the foremost technique for qualitative and quantitative lipidomics analyses. Glycerophospholipids and sphingolipids, collectively termed polar lipids, are commonly investigated by hyphenated liquid chromatography-mass spectrometry (LC-MS) techniques that reduce aggregation effects and provide a greater dynamic range of detection sensitivity compared to shotgun lipidomics. However, automatic polar lipid identification is hindered by several isobaric and isomer mass overlaps, which cause software programs to often fail to correctly annotate the lipid species. In the present paper, a buffer modification workflow based on the use of labeled and unlabeled acetate ions in the chromatographic buffers was optimized by Box-Behnken design of the experiments and applied to the characterization of phosphocholine-containing lipids in human plasma samples. The contemporary generation of [M + CH3COO]-, [M + CD3COO]-, and [M - CH3]- coupled with a dedicated data processing workflow, which was specifically set up on Compound Discoverer software, allowed us to correctly determine adduct composition, molecular formulas, and grouping, as well as granting a lower false-positive rate and streamlining the manual validation step compared to commonly employed lipidomics platforms. The proposed workflow represents a robust yet easier alternative to the existing approaches for improving lipid annotation, as it does not require extensive sample pretreatment or prior isotopic enrichment or derivatization.

Profiling and quantitative analysis of underivatized fatty acids in Chlorella vulgaris microalgae by liquid chromatography-high resolution mass spectrometry.

Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost-effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n-hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high-resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78-100%), intra- and interday relative standard deviations (<10 and <9%, respectively) and linearity (R2  > 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids.

A rapid and innovative extraction and enrichment method for the metaproteomic characterization of dissolved organic matter in groundwater samples.

Metaproteomic analysis of aquifer systems provides valuable information on the microbial populations, their influence on drinking water quality, and the effect on human health. In the present paper, an extraction and enrichment method by C18 extra-wide pore cartridge was developed, optimized, and applied for the first time to the metaproteomic characterization of dissolved organic matter in groundwater samples. In particular, three elution procedures were tested and compared on water spiked with a yeast protein extract to maximize the recovery of proteins from a complex matrix. The maximum protein recovery was obtained by the use of two sequential elution buffers, one employing a denaturing agent and the other one containing an acidified organic solvent. A comprehensive metaproteomic analysis of the dissolved organic matter of groundwater was then performed by nano-high performance liquid chromatography coupled to high-resolution mass spectrometry. A total of 239 proteins was identified; in agreement with the current knowledge on proteins in aquifer systems, most identified sequences derived from bacteria, protobacteria, and ciliates. The paper is the first metaproteomic study applied to groundwater samples with particular emphasis on the need for sample pretreatment to obtain comprehensive information on the proteome in dissolved organic matter.

Identification and Quantification of Polycyclic Aromatic Hydrocarbons in Polyhydroxyalkanoates Produced from Mixed Microbial Cultures and Municipal Organic Wastes at Pilot Scale.

Polyhydroxyalkanoates (PHAs) are well-known biodegradable plastics produced by various bacterial strains, whose major drawback is constituted by the high cost of their synthesis. Producing PHAs from mixed microbial cultures and employing organic wastes as a carbon source allows us to both reduce cost and valorize available renewable resources, such as food waste and sewage sludge. However, different types of pollutants, originally contained in organic matrices, could persist into the final product, thus compromising their safety. In this work, the exploitation of municipal wastes for PHA production is evaluated from the environmental and health safety aspect by determining the presence of polycyclic aromatic hydrocarbons (PAHs) in both commercial and waste-based PHA samples. Quantification of PAHs by gas chromatography-mass spectrometry on 24 PHA samples obtained in different conditions showed very low contamination levels, in the range of ppb to a few ppm. Moreover, the contaminant content seems to be dependent on the type of PHA stabilization and extraction, but independent from the type of feedstock. Commercial PHA derived from crops, selected for comparison, showed PAH content comparable to that detected in PHAs derived from organic fraction of municipal solid waste. Although there is no specific regulation on PAH maximum levels in PHAs, detected concentrations were consistently lower than threshold limit values set by regulation and guidelines for similar materials and/or applications. This suggests that the use of organic waste as substrate for PHA production is safe for both the human health and the environment.

High-Resolution Mass Spectrometry and Chemometrics for the Detailed Characterization of Short Endogenous Peptides in Milk By-Products.

The process of cheese-making has long been part of human food culture and nowadays dairy represents a large sector of the food industry. Being the main byproduct of cheese-making, the revalorization of milk whey is nowadays one of the primary goals in alignment with the principles of the circular economy. In the present paper, a deep and detailed investigation of short endogenous peptides in milk and its byproducts (whole whey, skimmed whey, and whey permeate) was carried out by high-resolution mass spectrometry, with a dedicated suspect screening data acquisition and data analysis approach. A total of 79 short peptides was tentatively identified, including several sequences already known for their exerted biological activities. An unsupervised chemometric approach was then employed for highlighting the differences in the short peptide content among the four sets of samples. Whole and skimmed whey showed not merely a higher content of short bioactive peptides compared to whole milk, but also a peculiar composition of peptides that are likely generated during the process of cheese-making. The results clearly demonstrate that whey represents a valuable source of bioactive compounds and that the set-up of processes of revalorization of milk byproducts is a promising path in the obtention of high revenue-generating products from dairy industrial waste.

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity.

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 µg mL-1). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 µg mL-1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.

Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics.

Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+. The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.

Untargeted Characterization of Chestnut (Castanea sativa Mill.) Shell Polyphenol Extract: A Valued Bioresource for Prostate Cancer Cell Growth Inhibition.

Chestnut seeds are used for fresh consumption and for the industrial preparation of derivatives, such as chestnut flour. During industrial processing, large amounts of by-products are generally produced, such as leaves, flowers, shells and burs. In the present study, chestnut shells were extracted by boiling water in order to obtain polyphenol-rich extracts. Moreover, for the removal or non-phenolic compounds, a separation by preparative reverse phase chromatography in ten fractions was carried out. The richest fractions in terms of phenolic content were characterized by means of untargeted high-resolution mass spectrometric analysis together with a dedicated and customized data processing workflow. A total of 243 flavonoids, phenolic acids, proanthocyanidins and ellagitannins were tentatively identified in the five richest fractions. Due its high phenolic content (450.03 µg GAE per mg of fraction), one tumor cell line (DU 145) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of fraction 3 dry extract for 24, 48 and 72 h. Moreover, for DU 145 cell lines, increase of apoptotic cells and perturbation of cell cycle was demonstrated for the same extract. Those outcomes suggest that chestnut industrial by-products could be potentially employed as a source of bioresources.

Development of an Analytical Method for the Metaproteomic Investigation of Bioaerosol from Work Environments.

The metaproteomic analysis of air particulate matter provides valuable information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters is developed. Different protein extraction procedures are tested to select the best extraction protocol based on protein recovery. The optimized method is tested for the extraction of proteins from spores of ubiquitous bacteria species and used for the metaproteomic characterization of filters from three work environments. In particular, ambient aerosol samples are collected in a composting plant, in a wastewater treatment plant, and in an agricultural holding. A total of 179, 15, 205, and 444 proteins are identified in composting plant, wastewater treatment plant, and agricultural holding, (cow stable and blending plant), respectively. In agreement with the major categories of primary biological aerosol particles, all identified proteins originated primarily from fungi, bacteria, and plants. The paper is the first metaproteomic study applied to bioaerosol samples collected in occupationally relevant environmental sites and, even though not aimed at monitoring the risk exposure of workers, it provides information on the possible exposure in the working environmental sites.

Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine.

Short peptide sequences represent emerging analytes in a variety of fields, including biomarker discovery, but also a well-known analytical challenge in complex matrices, due to the low abundance, extensive suppression during MS analysis, and lack of workflows, as they cannot be identified by ordinary peptidomics strategies and coverage is extremely limited by metabolomics as well. In this context, in this work, a solid phase extraction method was developed for the cleanup and enrichment of dipeptides, tripeptides, and tetrapeptides in urine using graphitized carbon black Carbograph 4 as the sorbent. The method was first developed on analytical standards spiked in urine, with recoveries in the range of 60-100%. Then the method was applied to urine samples from healthy volunteers. The enriched urine samples were analyzed by ultrahigh performance liquid chromatography (UHPLC) using an orthogonal strategy in which both a reversed phase (RP) C18 column and a zwitterionic hydrophilic interaction liquid chromatography (HILIC) column were used, for better coverage of peptide polarity and improved detection of peptides. High-resolution mass spectra were acquired in data-dependent mode using a suspect screening strategy with inclusion list; peptides were identified by a semiautomated workflow for feature extraction, candidate mass filtering, and MS/MS spectra comparison with in silico mass spectra. The complementarity of the orthogonal separation strategy was confirmed by peptide identification, resulting in 101 peptides identified from the RP runs and 111 peptides from the HILIC runs, with 60 common identifications. The method is applicable to both hydrophobic and hydrophilic peptides. Peptides were stable over 2 h after collection and protease inhibitors were not necessary, as no formation of artifacts was observed.

Liposome protein corona characterization as a new approach in nanomedicine.

This trends article describes the analytical approaches for the in-depth characterization of the protein corona on liposome nanoparticles. In particular, examples since 2014 are summarized according to the analytical approach. Traditional protein corona characterizations from in vitro static experiments are provided along with the newly introduced experimental setups for characterization of the protein corona by in vitro dynamic and in vivo studies. Additionally, a special attention is also devoted to the need for introduction of new experimental workflows for characterization of a much wider array of biomolecules. In the most recent years, an extension of the protein corona concept to the biomolecular corona was introduced, and the analytical targets are no longer restricted to proteins, but to other important biomolecules as well, as they can potentially affect the biodistribution and interaction of nanoparticles with the biological systems. The few recent examples in this field are discussed for the characterization of metabolites and lipids in the biomolecular corona with examples, also extending the discussion from liposome to other types of nanoparticles. A final discussion is provided on the potential key role of the most recent omics approaches in the study of the nano-bio interface, with an overview on top-down proteomics, which allows a better elucidation of proteoforms, and on lipidomics and metabolomics, which allow a comprehensive untargeted characterization of lipids and metabolites, respectively. Graphical abstract.