In-gel protein digestion using acidic methanol produces a highly selective methylation of glutamic acid residues.

Mass-tolerant open search methods allow the high-throughput analysis of modified peptides by mass spectrometry. These techniques have paved the way to unbiased analysis of post-translational modifications in biological contexts, as well as of chemical modifications produced during the manipulation of protein samples. In this work, we have analyzed in-depth a wide variety of samples of different biological origin, including cells, extracellular vesicles, secretomes, centrosomes and tissue preparations, using Comet-ReCom, a recently improved version of the open search engine Comet-PTM. Our results demonstrate that glutamic acid residues undergo intensive methyl esterification when protein digestion is performed using in-gel techniques, but not using gel-free approaches. This effect was highly specific to Glu and was not found for other methylable residues such as Asp.

Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation.

Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine-histidine-purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

iSanXoT: A standalone application for the integrative analysis of mass spectrometry-based quantitative proteomics data.

Many bioinformatics tools are available for the quantitative analysis of proteomics experiments. Most of these tools use a dedicated statistical model to derive absolute quantitative protein values from mass spectrometry (MS) data. Here, we present iSanXoT, a standalone application that processes relative abundances between MS signals and then integrates them sequentially to upper levels using the previously published Generic Integration Algorithm (GIA). iSanXoT offers unique capabilities that complement conventional quantitative software applications, including statistical weighting and independent modeling of error distributions in each integration, aggregation of technical or biological replicates, quantification of posttranslational modifications, and analysis of coordinated protein behavior. iSanXoT is a standalone, user-friendly application that accepts output from popular proteomics pipelines and enables unrestricted creation of quantification workflows and fully customizable reports that can be reused across projects or shared among users. Numerous publications attest the successful application of diverse integrative workflows constructed using the GIA for the analysis of high-throughput quantitative proteomics experiments. iSanXoT has been tested with the main operating systems. Download links for the corresponding distributions are available at https://github.com/CNIC-Proteomics/iSanXoT/releases.

Oxidative Post-translational Protein Modifications upon Ischemia/Reperfusion Injury.

While reperfusion, or restoration of coronary blood flow in acute myocardial infarction, is a requisite for myocardial salvage, it can paradoxically induce a specific damage known as ischemia/reperfusion (I/R) injury. Our understanding of the precise pathophysiological molecular alterations leading to I/R remains limited. In this study, we conducted a comprehensive and unbiased time-course analysis of post-translational modifications (PTMs) in the post-reperfused myocardium of two different animal models (pig and mouse) and evaluated the effect of two different cardioprotective therapies (ischemic preconditioning and neutrophil depletion). In pigs, a first wave of irreversible oxidative damage was observed at the earliest reperfusion time (20 min), impacting proteins essential for cardiac contraction. A second wave, characterized by irreversible oxidation on different residues and reversible Cys oxidation, occurred at late stages (6-12 h), affecting mitochondrial, sarcomere, and inflammation-related proteins. Ischemic preconditioning mitigated the I/R damage caused by the late oxidative wave. In the mouse model, the two-phase pattern of oxidative damage was replicated, and neutrophil depletion mitigated the late wave of I/R-related damage by preventing both Cys reversible oxidation and irreversible oxidation. Altogether, these data identify protein PTMs occurring late after reperfusion as an actionable therapeutic target to reduce the impact of I/R injury.

Immune synapse formation promotes lipid peroxidation and MHC-I upregulation in licensed dendritic cells for efficient priming of CD8+ T cells.

Antigen cognate dendritic cell (DC)-T cell synaptic interactions drive activation of T cells and instruct DCs. Upon receiving CD4+ T cell help, post-synaptic DCs (psDCs) are licensed to generate CD8+ T cell responses. However, the cellular and molecular mechanisms that enable psDCs licensing remain unclear. Here, we describe that antigen presentation induces an upregulation of MHC-I protein molecules and increased lipid peroxidation on psDCs in vitro and in vivo. We also show that these events mediate DC licensing. In addition, psDC adoptive transfer enhances pathogen-specific CD8+ T responses and protects mice from infection in a CD8+ T cell-dependent manner. Conversely, depletion of psDCs in vivo abrogates antigen-specific CD8+ T cell responses during immunization. Together, our data show that psDCs enable CD8+ T cell responses in vivo during vaccination and reveal crucial molecular events underlying psDC licensing.

Quantification of Farnesylated Progerin in Hutchinson-Gilford Progeria Patient Cells by Mass Spectrometry.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal disorder characterized by premature aging and death at a median age of 14.5 years. The most common cause of HGPS (affecting circa 90% of patients) is a de novo heterozygous synonymous single-base substitution (c.1824C>T; p.G608G) in the LMNA gene that results in the accumulation of progerin, an aberrant form of lamin A that, unlike mature lamin A, remains permanently farnesylated. The ratio of progerin to mature lamin A correlates with disease severity in HGPS patients, and can be used to assess the effectiveness of therapies aimed at lessening aberrant splicing or progerin farnesylation. We recently showed that the endogenous content of lamin A and progerin can be measured by mass spectrometry (MS), providing an alternative to immunological methods, which lack the necessary specificity and quantitative accuracy. Here, we present the first non-immunological method that reliably quantifies the levels of wild-type lamin A and farnesylated progerin in cells from HGPS patients. This method, which is based on a targeted MS approach and the use of isotope-labeled internal standards, could be applied in ongoing clinical trials evaluating the efficacy of drugs that inhibit progerin farnesylation.

Extracellular vesicles from Listeria monocytogenes-infected dendritic cells alert the innate immune response.

Communication through cell-cell contacts and extracellular vesicles (EVs) enables immune cells to coordinate their responses against diverse types of pathogens. The function exerted by EVs in this context depends on the proteins and nucleic acids loaded into EVs, which elicit specific responses involved in the resolution of infection. Several mechanisms control protein and nucleic acid loading into EVs; in this regard, acetylation has been described as a mechanism of cellular retention during protein sorting to exosomes. HDAC6 is a deacetylase involved in the control of cytoskeleton trafficking, organelle polarity and cell migration, defense against Listeria monocytogenes (Lm) infection and other immune related functions. Here, we show that the protein content of dendritic cells (DCs) and their secreted EVs (DEVs) vary during Lm infection, is enriched in proteins related to antiviral functions compared to non-infected cells and depends on HDAC6 expression. Analyses of the post-translational modifications revealed an alteration of the acetylation and ubiquitination profiles upon Lm infection both in DC lysates and DEVs. Functionally, EVs derived from infected DCs upregulate anti-pathogenic genes (e.g. inflammatory cytokines) in recipient immature DCs, which translated into protection from subsequent infection with vaccinia virus. Interestingly, absence of Listeriolysin O in Lm prevents DEVs from inducing this anti-viral state. In summary, these data underscore a new mechanism of communication between bacteria-infected DC during infection as they alert neighboring, uninfected DCs to promote antiviral responses.

Specialized Proresolving Mediators Protect Against Experimental Autoimmune Myocarditis by Modulating Ca2+ Handling and NRF2 Activation.

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum-adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

Expression of spidroin proteins in the silk glands of golden orb-weaver spiders.

The expression of spidroins in the major ampullate, minor ampullate, flagelliform, and tubuliform silk glands of Trichonephila clavipes spiders was analyzed using proteomics analysis techniques. Spidroin peptides were identified and assigned to different gene products based on sequence concurrence when compared with the whole genome of the spider. It was found that only a relatively low proportion of the spidroin genes are expressed as proteins in any of the studied glands. In addition, the expression of spidroin genes in different glands presents a wide range of patterns, with some spidroins being found in a single gland exclusively, while others appear in the content of several glands. The combination of precise genomics, proteomics, microstructural, and mechanical data provides new insights both on the design principles of these materials and how these principles might be translated for the production of high-performance bioinspired artificial fibers.

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

AIMS: Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. METHODS AND RESULTS: Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. CONCLUSION: ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.

Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production.

Vaccine therapies based on virus-like particles (VLPs) are currently in the spotlight due to their potential for generating high immunogenic responses while presenting fewer side effects than conventional vaccines. These self-assembled nanostructures resemble the native conformation of the virus but lack genetic material. They are becoming a promising platform for vaccine candidates against several diseases due to the ability of modifying their membrane with antigens from different viruses. The coproduction of extracellular vesicles (EVs) when producing VLPs is a key phenomenon currently still under study. In order to characterize this extracellular environment, a quantitative proteomics approach has been carried out. Three conditions were studied: non-transfected, transfected with an empty plasmid as control, and transfected with a plasmid coding for HIV-1 Gag polyprotein. A shift in EV biogenesis has been detected upon transfection, changing the production from large to small EVs. Another remarkable trait found was the presence of DNA being secreted within vesicles smaller than 200 nm. Studying the protein profile of these biological nanocarriers, it was observed that EVs were reflecting an overall energy homeostasis disruption via mitochondrial protein deregulation. Also, immunomodulatory proteins like ITGB1, ENO3, and PRDX5 were identified and quantified in VLP and EV fractions. These findings provide insight on the nature of the VLP extracellular environment defining the characteristics and protein profile of EVs, with potential to develop new downstream separation strategies or using them as adjuvants in viral therapies.

Identification of common cardiometabolic alterations and deregulated pathways in mouse and pig models of aging.

Aging is the main risk factor for cardiovascular and metabolic diseases, which have become a global concern as the world population ages. These diseases and the aging process are exacerbated in Hutchinson-Gilford progeria syndrome (HGPS or progeria). Here, we evaluated the cardiometabolic disease in animal models of premature and normal aging with the aim of identifying alterations that are shared or specific to each condition. Despite differences in body composition and metabolic markers, prematurely and normally aging mice developed heart failure and similar cardiac electrical abnormalities. High-throughput proteomics of the hearts of progeric and normally aged mice revealed altered protein oxidation and glycation, as well as dysregulated pathways regulating energy metabolism, proteostasis, gene expression, and cardiac muscle contraction. These results were corroborated in the hearts of progeric pigs, underscoring the translational potential of our findings, which could help in the design of strategies to prevent or slow age-related cardiometabolic disease.

The Immunomodulatory Signature of Extracellular Vesicles From Cardiosphere-Derived Cells: A Proteomic and miRNA Profiling.

Experimental data demonstrated that the regenerative potential and immunomodulatory capacity of cardiosphere-derived cells (CDCs) is mediated by paracrine mechanisms. In this process, extracellular vesicles derived from CDCs (EV-CDCs) are key mediators of their therapeutic effect. Considering the future applicability of these vesicles in human diseases, an accurate preclinical-to-clinical translation is needed, as well as an exhaustive molecular characterization of animal-derived therapeutic products. Based on that, the main goal of this study was to perform a comprehensive characterization of proteins and miRNAs in extracellular vesicles from porcine CDCs as a clinically relevant animal model. The analysis was performed by identification and quantification of proteins and miRNA expression profiles. Our results revealed the presence of clusters of immune-related and cardiac-related molecular biomarkers in EV-CDCs. Additionally, considering that priming stem cells with inflammatory stimuli may increase the therapeutic potential of released vesicles, here we studied the dynamic changes that occur in the extracellular vesicles from IFNγ-primed CDCs. These analyses detected statistically significant changes in several miRNAs and proteins. Notably, the increase in interleukin 6 (IL6) protein, as well as the increase in mir-125b (that targets IL6 receptor) was especially relevant. These results suggest a potential involvement of EV-CDCs in the regulation of the IL6/IL6R axis, with implications in inflammatory-mediated diseases.

Complement C5 Protein as a Marker of Subclinical Atherosclerosis.

BACKGROUND: The mechanisms underlying early atherosclerotic plaque formation are not completely understood. Moreover, plasma biomarkers of subclinical atherosclerosis are lacking. OBJECTIVES: The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets. METHODS: The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media and intima layers) was analyzed by deep quantitative multiplexed proteomics. Further analysis was performed by Western blot, immunohistochemistry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (>2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts (PESA [Progression of Early Subclinical Atherosclerosis] [n = 360] and NEFRONA [National Observatory of Atherosclerosis in Nephrology] [n = 394]). RESULTS: Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification. CONCLUSIONS: Activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinical atherosclerosis.

Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production.

The production of virus-like particles (VLPs) has gained importance over the last few years owing to the benefits they provide compared to conventional vaccines. The biopharmaceutical industry is currently searching for safer candidates based on VLPs for new and existing vaccines and implementing new methods of manufacturing, thus allowing a more sustainable, effective, and species-specific production. Despite achieving lower yields compared to traditional platforms, the use of mammalian cells provides the right post-translational modifications, and consequently, the intensification of bioprocesses using mammalian cell platforms has become a matter of pressing concern. One of the methods subjected to intensification is transient gene expression, which has been proven to be highly effective regarding VLP production for preclinical or even clinical trials. In this work, a multiplexed quantitative proteomic approach has been applied to study the molecular characteristics of HEK293 cell cultures when growing at cell densities higher than 4 × 106 cells/mL and to study the effects related to cell transfection and VLP production. The obtained results revealed a set of functional and metabolic profiles of HEK293 under these three different conditions that allowed the identification of physiological bottlenecks regarding VLP production. Regarding the cell density effect, molecular alterations in the cell biology were proposed to help explain the difficulty for the cells to be transfected at higher densities. In addition, an overall disruption of cellular homeostasis after transfection was observed based on altered biological processes, and after identifying potential pathways liable to be optimized via metabolic engineering, different solutions were proposed to improve VLP production.

A Proteomics Signature of Mild Hypospadias: A Pilot Study.

Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients. Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples. Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias. Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation.

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1ß, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1ß, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1ß stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1ß-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1ß/IL-17 axis.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation.

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

APOA1 oxidation is associated to dysfunctional high-density lipoproteins in human abdominal aortic aneurysm.

BACKGROUND: High-density lipoproteins (HDL) are a complex mixture of lipids and proteins with vasculoprotective properties. However, HDL components could suffer post-translational modifications (PTMs) under pathological conditions, leading to dysfunctional HDL. We studied whether HDL are modified in abdominal aortic aneurysm (AAA) and the effect on HDL functionality. METHODS: HDL were isolated by ultracentrifugation from AAA tissue (HDL-T) and from plasma of healthy volunteers and then incubated with AAA tissue-conditioned medium (HDL-AAA CM). PTMs from these particles were characterized using Comet-PTM. The ability of HDL-AAA CM for promoting cholesterol efflux was determined ex vivo and in vivo by using J774A.1 [3H]cholesterol-labeled mouse macrophages and after injecting [3H]cholesterol-labeled mouse macrophages and HDL into the peritoneal cavity of wild-type C57BL/6 mice, respectively. Trp50 and Trp108 oxidized forms of APOA1 in HDL incubated with conditioned-medium of activated neutrophils and in plasma of AAA patients and controls were measured by targeted parallel reaction monitoring. FINDINGS: Oxidation was the most prevalent PTM in apolipoproteins, particularly in APOA1. Trp50 and Trp108 in APOA1 were the residues most clearly affected by oxidation in HDL-T and in HDL-AAA CM, when compared to their controls. In addition, cholesterol efflux was decreased in macrophages incubated with HDL-AAA CM in vitro and a decreased macrophage-to-serum reverse cholesterol transport was also observed in mice injected with HDL-AAA CM. Finally, both oxidized Trp50 and Trp108 forms of APOA1 were increased in HDL incubated with conditioned-medium of activated neutrophils and in plasma of AAA patients in relation to controls. INTERPRETATION: Oxidative modifications of HDL present in AAA tissue and plasma were closely associated with the loss of vasculoprotective properties of HDL in AAA. FUND: MINECO, ISCiii-FEDER, CIBERDEM, CIBERCV and LA CAIXA.