Adipocyte p53 coordinates the response to intermittent fasting by regulating adipose tissue immune cell landscape.

In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans.

Structural specializations of the sperm tail.

Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.

Detection and Characterization of a De Novo Alu Retrotransposition Event Causing NKX2-1-Related Disorder.

BACKGROUND: Heterozygous NKX2-1 loss-of-function variants cause combinations of hyperkinetic movement disorders (MDs, particularly childhood-onset chorea), pulmonary dysfunction, and hypothyroidism. Mobile element insertions (MEIs) are potential disease-causing structural variants whose detection in routine diagnostics remains challenging. OBJECTIVE: To establish the molecular diagnosis of two first-degree relatives with clinically suspected NKX2-1-related disorder who had negative NKX2-1 Sanger (SS), whole-exome (WES), and whole-genome (WGS) sequencing. METHODS: The proband's WES was analyzed for MEIs. A candidate MEI in NKX2-1 underwent optimized SS after plasmid cloning. Functional studies exploring NKX2-1 haploinsufficiency at RNA and protein levels were performed. RESULTS: A 347-bp AluYa5 insertion with a 65-bp poly-A tail followed by a 16-bp duplication of the pre-insertion wild-type sequence in exon 3 of NKX2-1 (ENST00000354822.7:c.556_557insAlu541_556dup) segregated with the disease phenotype. CONCLUSIONS: We identified a de novo exonic AluYa5 insertion causing NKX2-1-related disorder in SS/WES/WGS-negative cases, suggesting that MEI analysis of short-read sequencing data or targeted long-read sequencing could unmask the molecular diagnosis of unsolved MD cases. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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-.

Complementary omics strategies to dissect p53 signaling networks under nutrient stress.

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo.

For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes' synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or "proteoDNAsome", which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity.

In-cell structures of conserved supramolecular protein arrays at the mitochondria-cytoskeleton interface in mammalian sperm.

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

ß-RA Targets Mitochondrial Metabolism and Adipogenesis, Leading to Therapeutic Benefits against CoQ Deficiency and Age-Related Overweight.

Primary mitochondrial diseases are caused by mutations in mitochondrial or nuclear genes, leading to the abnormal function of specific mitochondrial pathways. Mitochondrial dysfunction is also a secondary event in more common pathophysiological conditions, such as obesity and metabolic syndrome. In both cases, the improvement and management of mitochondrial homeostasis remain challenging. Here, we show that beta-resorcylic acid (ß-RA), which is a natural phenolic compound, competed in vivo with 4-hydroxybenzoic acid, which is the natural precursor of coenzyme Q biosynthesis. This led to a decrease in demethoxyubiquinone, which is an intermediate metabolite of CoQ biosynthesis that is abnormally accumulated in Coq9R239X mice. As a consequence, ß-RA rescued the phenotype of Coq9R239X mice, which is a model of primary mitochondrial encephalopathy. Moreover, we observed that long-term treatment with ß-RA also reduced the size and content of the white adipose tissue (WAT) that is normally accumulated during aging in wild-type mice, leading to the prevention of hepatic steatosis and an increase in survival at the elderly stage of life. The reduction in WAT content was due to a decrease in adipogenesis, an adaptation of the mitochondrial proteome in the kidneys, and stimulation of glycolysis and acetyl-CoA metabolism. Therefore, our results demonstrate that ß-RA acted through different cellular mechanisms, with effects on mitochondrial metabolism; as such, it may be used for the treatment of primary coenzyme Q deficiency, overweight, and hepatic steatosis.

A serum proteome signature to predict mortality in severe COVID-19 patients.

Here, we recorded serum proteome profiles of 33 severe COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later time points. With the aim to predict treatment outcome, we focused on serum proteins different in abundance between the group of survivors and non-survivors. We observed that a small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, histidine-rich glycoprotein, and KNG1 were all more abundant in the survivors. The family of inter-α-trypsin inhibitors, ITIH1, ITIH2, ITIH3, and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also showed opposite trends in protein abundance during disease progression. We defined an optimal panel of nine proteins for mortality risk assessment. The prediction power of this mortality risk panel was evaluated against two recent COVID-19 serum proteomics studies on independent cohorts measured in other laboratories in different countries and observed to perform very well in predicting mortality also in these cohorts. This panel may not be unique for COVID-19 as some of the proteins in the panel have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria.

Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle.

Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides. The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices.

Identification and Antimicrobial Activity of Medium-Sized and Short Peptides from Yellowfin Tuna (Thunnus albacares) Simulated Gastrointestinal Digestion.

Due to the rapidly increasing resistance to conventional antibiotics, antimicrobial peptides are emerging as promising novel drug candidates. In this study, peptide fragments were obtained from yellowfin tuna muscle by simulated gastrointestinal digestion, and their antimicrobial activity towards Gram-positive and Gram-negative bacteria was investigated. In particular, the antimicrobial activity of both medium- and short-sized peptides was investigated by using two dedicated approaches. Medium-sized peptides were purified by solid phase extraction on C18, while short peptides were purified thanks to a graphitized carbon black sorbent. For medium-sized peptide characterization, a peptidomic strategy based on shotgun proteomics analysis was employed, and identification was achieved by matching protein sequence database by homology, as yellowfin tuna is a non-model organism, leading to the identification of 403 peptides. As for short peptide sequences, an untargeted suspect screening approach was carried out by means of an inclusion list presenting the exact mass to charge ratios (m/z) values for all di-, tri- and tetrapeptides. In total, 572 short sequences were identified thanks to a customized workflow dedicated to short peptide analysis implemented on Compound Discoverer software.

A protein corona sensor array detects breast and prostate cancers.

Following exposure to human plasma (HP), nanoparticles (NPs) are coated with a biomolecular layer referred to as a protein corona. We recently revealed that characterizing the protein coronas of various NPs may provide a unique opportunity for cancer identification and discrimination. In other words, protein corona profiles of several NPs, when being analyzed using classifiers, would provide a unique "fingerprint" for each type of disease. Here, we probed the capacity of the protein corona for the identification and discrimination of breast and prostate cancer patients from healthy individuals. Using three lipid NP formulations with distinct physical-chemical properties as a cross-reactive sensor array and a supervised random forest classifier, we identified a set of proteins that showed a significant difference in cancer patients and control subjects. Our data show that many of the corona proteins with the highest discrimination ability between oncological patients and healthy individuals are related to cellular and molecular aspects of breast and prostate cancers.

Personalized Graphene Oxide-Protein Corona in the Human Plasma of Pancreatic Cancer Patients.

The protein corona (PC) that forms around nanomaterials upon exposure to human biofluids (e.g., serum, plasma, cerebral spinal fluid etc.) is personalized, i.e., it depends on alterations of the human proteome as those occurring in several cancer types. This may relevant for early cancer detection when changes in concentration of typical biomarkers are often too low to be detected by blood tests. Among nanomaterials under development for in vitro diagnostic (IVD) testing, Graphene Oxide (GO) is regarded as one of the most promising ones due to its intrinsic properties and peculiar behavior in biological environments. While recent studies have explored the binding of single proteins to GO nanoflakes, unexplored variables (e.g., GO lateral size and protein concentration) leading to formation of GO-PC in human plasma (HP) have only marginally addressed so far. In this work, we studied the PC that forms around GO nanoflakes of different lateral sizes (100, 300, and 750 nm) upon exposure to HP at several dilution factors which extend over three orders of magnitude from 1 (i.e., undiluted HP) to 103. HP was collected from 20 subjects, half of them being healthy donors and half of them diagnosed with pancreatic ductal adenocarcinoma (PDAC) a lethal malignancy with poor prognosis and very low 5-year survival rate after diagnosis. By dynamic light scattering (DLS), electrophoretic light scattering (ELS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nano liquid chromatography tandem mass spectrometry (nano-LC MS/MS) experiments we show that the lateral size of GO has a minor impact, if any, on PC composition. On the other side, protein concentration strongly affects PC of GO nanoflakes. In particular, we were able to set dilution factor of HP in a way that maximizes the personalization of PC, i.e., the alteration in the protein profile of GO nanoflakes between cancer vs. non-cancer patients. We believe that this study shall contribute to a deeper understanding of the interactions among GO and HP, thus paving the way for the development of IVD tools to be used at every step of the patient pathway, from prognosis, screening, diagnosis to monitoring the progression of disease.

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.

Does the protein corona take over the selectivity of molecularly imprinted nanoparticles? The biological challenges to recognition.

"Plastic antibodies" are nano-sized biomimetics prepared by the molecular imprinting technology, which have the robustness of polymers, but specificity and selectivity alike natural receptors making them ideal for analytical uses. The current challenge is to translate plastic antibodies to in vivo applications for diagnosis, drug delivery, theranostic, therefore it is crucial to evaluate the effect of the biological sample complexity on the selectivity and the formation of protein corona (PCs), which ultimately dictate the fate of circulating nanoparticles. A set (n = 4) of plastic antibodies (nanoMIPs) against different proteins was prepared. Quantitative (iBAC) shotgun proteomics permitted to define the PC composition of nanoMIPs in human plasma, the relative protein abundances, the correlation between PC and the plasma dilution. NanoMIPs showed >200 proteins PC, while ~150 proteins were found on controls, suggesting the imprinting process influences the nanoparticle's structure hence the protein uptake. NanoMIPs and controls shared the 44% of the PC, but PC iBAQ values on nanoMIPs were 10-100 times higher than controls, suggesting PC/nanoMIPs interactions were far stronger than PC/non imprinted particles. PCs were richer in small proteins and in immunoglobulins, indicating a defensive response, while the selectivity was negatively challenged in the crowded plasma sample. SIGNIFICANCE: The formation and the composition of the protein corona (PC) is key to decide the fate of nanoparticles when in vivo, therefore there is the strong need to study the composition of the PC. To enable and to support the translation of the use of plastic antibodies (nanoMIPs), prepared by means of the molecular imprinting technique, to the clinical practice and to in vivo uses, the present work evaluates the effects of the complexity of the biological sample (plasma) on nanoMIPs composed of highly crosslinked polyacrylamide and acrylamide derivatives. Proteomic study offers an in depth insight of the protein corona formed in plasma on nanoMIPs. A set of nanoMIPs synthesized and raised to recognize either small or large proteins was tested. The selection abilities of the nanoMIPs when placed in plasma at different dilutions was studied. Quantitative shotgun proteomics allowed to define the composition of the formed protein corona (PC) enabling to detail the protein compositions, the relative abundances, its correlation to the biological sample composition and the correlation between PC and nanoMIP's imprinted template. In plasma, all the nanoMIPs gained a PC composed of more than 200 proteins. Type of protein recruited for the corona, molecular weight and abundance in the PC were studied. The PC on the nanoMIPs appeared to be driven by the protein composition of the plasma, while the template protein, towards which a nanoMIP was imprinted and that was proven to have high affinity for, did not influence the PC.

Peptidomic Approach for the Identification of Peptides with Potential Antioxidant and Anti-Hyperthensive Effects Derived From Asparagus By-Products.

Asparagus waste represents products of great interest since many compounds with high biological value are located in the lower portion of the spears. The extraction of bioactive compounds from asparagus by-products is therefore crucial for the purpose of adding value to these by-products. In this paper, bioactive peptides from asparagus waste were extracted, digested, purified and identified. In particular, Alcalase® was chosen as the enzyme to use to obtain protein hydrolysate due to its low cost and, consequently, the possibility of implementing the method on a large scale. In order to simplify the peptide extract to reach better identification, the hydrolysate was fractionated by reversed-phase chromatography in 10 fractions. Two tests were carried out for antioxidant activity (ABTS-DPPH) and one for antihypertensive activity (ACE). Fractions with a higher bioactivity score were identified by peptidomics technologies and screened for bioactivity with the use of bioinformatics. For ACE-inhibitor activity, two peptides were synthetized, PDWFLLL and ASQSIWLPGWL, which provided an EC50 value of 1.76 µmol L-1 and 4.02 µmol L-1, respectively. For the antioxidant activity, by DPPH assay, MLLFPM exhibited the lowest EC50 value at 4.14 µmol L-1, followed by FIARNFLLGW and FAPVPFDF with EC50 values of 6.76 µmol L-1 and 10.01 µmol L-1, respectively. A validation of the five identified peptides was also carried out. The obtained results showed that peptides obtained from asparagus by-products are of interest for their biological activity and are suitable for being used as functional ingredients.

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.

The biomolecular corona of gold nanoparticles in a controlled microfluidic environment.

Nanoparticles (NPs) exposed to biological media are coated by proteins and other biomolecules forming a biomolecular corona (BC) on the particle surface. Recent studies have shown that shear stress as that created by laminar fluid flow generates more complex coronas with systematic changes in composition with respect to counterparts formed under static incubation. However, in most studies reported so far, dynamic environments have been produced by peristaltic pumps and comparing experimental results appears challenging. On the other side, generating shear stress by microfluidic devices could help to remove user variability and ensure better reproducibility of experimental data. This study was therefore aimed at exploring formation of NP-BC in a microfluidic environment. To this end, 100 nm gold nanoparticles and human plasma (HP) were used as models for nano-formulation and biological medium. We injected gold nanoparticles and HP in each of the islets of a remote-controlled microfluidic cartridge. Static incubation was used as a reference. BC-decorated NPs were thoroughly characterized by dynamic light scattering (DLS), micro-electrophoresis (ME), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS). By varying the incubation time from 30 s to 2.5 min we demonstrate that BC is already determined by the earliest exposure time point and does not appreciably evolve in time. DLS and ME results demonstrate that the BC formed in a microfluidic chip is thicker and more negatively charged than its counterpart formed under static incubation. SDS-PAGE and nano-LC MS/MS revealed that the incubation procedure had a major effect on BC composition. As an example, immunoglobulins are the most abundant plasma proteins of the BC generated in a microfluidic environment (relative protein abundance ∼30%), while tissue leakage proteins (relative protein abundance ∼26%) are the most enriched proteins when the BC is formed upon static incubation. Potential implications in emerging biomedical research arenas are discussed.

Effect of Glucose on Liposome-Plasma Protein Interactions: Relevance for the Physiological Response of Clinically Approved Liposomal Formulations.

Recently, the concept is emerging that the reduced success of nanoparticles in clinical practice is due to the adsorption of the "biomolecular corona (BC)," which alters their biological identity. Apart from protein variations, alterations in the human metabolome may change the BC decoration, which has poorly been addressed so far. Here, glucose is used as a model metabolite and how the interactions between liposomes (as a model nanoparticle) and plasma proteins are influenced by normal and diabetic sugar blood levels is explored. As model liposomes, Doxoves and Onivyde are used that are used for the treatment of breast and metastatic pancreatic cancer, respectively. It is shown that glucose does affect the structure and composition of BC. The biological effects of liposome-BC complexes are investigated in MCF 7 and MDA-MB-231 breast cancer cells for Doxoves and in pancreatic adenocarcinoma (PANC-1) and insulinoma (INS-1) cells for Onivyde. In the presence of glucose, the cellular toxicity of liposome-protein complexes and uptake by human monocytic THP1 cell line increases. These results demonstrate that alterations in glucose concentration, and more generally changes in the human metabolome, may play a fundamental role in the biological identity of liposomes and, consequently, on their in vivo physiological readouts including therapeutic efficacy.

Saliva as a source of new phosphopeptide biomarkers: Development of a comprehensive analytical method based on shotgun peptidomics.

The paper describes the development of an enrichment method for the analysis of the endogenous phosphopeptides in saliva. The method was based on magnetic solid phase extraction by a magnetic graphitized carbon black-TiO2 composite material and was developed considering different saliva pre-treatments, namely C18 solid phase extraction for purification, direct dilution in loading buffer or acetonitrile precipitation. The method was based on a shotgun proteomics workflow and the enriched peptide mixture was analysed by nanoHPLC and high resolution tandem mass spectrometry. Acetonitrile precipitation provided the best results, with up to 165 endogenous phosphopeptides identified in saliva samples from healthy individuals. The physico-chemical features of the identified endogenous phosphopeptides indicated that such peptides were large, hydrophilic and basic.