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Background: By analyzing the proteins which are the workhorses of biological systems, metaproteomics allows us to list the taxa present in any microbiota, monitor their relative biomass, and characterize the functioning of complex biological systems., Results: Here, we present a new strategy for rapidly determining the microbial community structure of a given sample and designing a customized protein sequence database to optimally exploit extensive tandem mass spectrometry data. This approach leverages the capabilities of the first generation of Quadrupole Orbitrap mass spectrometer incorporating an asymmetric track lossless (Astral) analyzer, offering rapid MS/MS scan speed and sensitivity. We took advantage of data-dependent acquisition and data-independent acquisition strategies using a peptide extract from a human fecal sample spiked with precise amounts of peptides from two reference bacteria., Conclusions: Our approach, which combines both acquisition methods, proves to be time-efficient while processing extensive generic databases and massive datasets, achieving a coverage of more than 122,000 unique peptides and 38,000 protein groups within a 30-min DIA run. This marks a significant departure from current state-of-the-art metaproteomics methodologies, resulting in broader coverage of the metabolic pathways governing the biological system. In combination, our strategy and the Astral mass analyzer represent a quantum leap in the functional analysis of microbiomes. Video Abstract., (© 2024. The Author(s).)

Introduction: Post translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy., Methods: A citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model., Results: We show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide., Conclusion: We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach., Competing Interests: Authors SK, RMP were employed by Proteome Sciences R & D GmbH & Co.KG. LD is a director and shareholder in Scancell Ltd. The remaining authors are employees of Scancell Ltd., (Copyright © 2022 Brentville, Symonds, Chua, Skinner, Daniels, Cook, Koncarevic, Martinez-Pinna, Shah, Choudhury, Vaghela, Weston, Al-Omari, Davis and Durrant.)

The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.

Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

To assess the potential association between TRX-1/PRX-1 and NADPH oxidase (Nox) activity in vivo and in vitro, TRX-1/PRX-1 levels were assessed by ELISA in 84 asymptomatic subjects with known phagocytic NADPH oxidase activity and carotid intima-media thickness (IMT). We found a positive correlation between TRX-1/PRX-1 and NADPH oxidase-dependent superoxide production (r=0.48 and 0.47; p<0.001 for both) and IMT (r=0.31 and 0.36; p<0.01 for both) adjusted by age and sex. Moreover, asymptomatic subjects with plaques have higher PRX-1 and TRX plasma levels (p<0.01 for both). These data were confirmed in a second study in which patients with carotid atherosclerosis showed higher PRX-1 and TRX plasma levels than healthy subjects (p<0.001 for both). In human atherosclerotic plaques, the NADPH oxidase subunit p22phox colocalized with TRX-1/PRX-1 in macrophages (immunohistochemistry). In monocytes and macrophages, phorbol 12-myristate 13-acetate (PMA) induced NADPH activation and TRX-1/PRX-1 release to the extracellular medium, with a concomitant decrease in their intracellular levels, which was reversed by the NADPH inhibitor apocynin (Western blot). In loss-of-function experiments, genetic silencing of the NADPH oxidase subunit Nox2 blocked PMA-induced intracellular TRX-1/PRX-1 downregulation in macrophages. Furthermore, the PMA-induced release of TRX-1/PRX-1 involves the modulation of their redox status and exosome-like vesicles. TRX-1/PRX-1 levels are associated with NADPH oxidase-activity in vivo and in vitro. These data could suggest a coordinated antioxidant response to oxidative stress in atherothrombosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Abdominal aortic aneurysm (AAA) evolution is unpredictable, and there is no therapy except surgery for patients with an aortic size> 5 cm (large AAA). We aimed to identify new potential biomarkers that could facilitate prognosis and treatment of patients with AAA. A differential quantitative proteomic analysis of plasma proteins was performed in AAA patients at different stages of evolution [small AAA (aortic size=3-5 cm) vs large AAA] using iTRAQ labelling, high-throughput nano-LC-MS/MS and a novel multi-layered statistical model. Among the proteins identified, ApoA-I was decreased in patients with large AAA compared to those with small AAA. These results were validated by ELISA on plasma samples from small (n=90) and large AAA (n=26) patients (150± 3 vs 133± 5 mg/dl, respectively, p< 0.001). ApoA-I levels strongly correlated with HDL-Cholesterol (HDL-C) concentration (r=0.9, p< 0.001) and showed a negative correlation with aortic size (r=-0.4, p< 0.01) and thrombus volume (r=-0.3, p< 0.01), which remained significant after adjusting for traditional risk factors. In a prospective study, HDL-C independently predicted aneurysmal growth rate in multiple linear regression analysis (n=122, p=0.008) and was inversely associated with need for surgical repair (Adjusted hazard ratio: 0.18, 95 % confidence interval: 0.04-0.74, p=0.018). In a nation-wide Danish registry, we found lower mean HDL-C concentration in large AAA patients (n=6,560) compared with patients with aorto-iliac occlusive disease (n=23,496) (0.89± 2.99 vs 1.59± 5.74 mmol/l, p< 0.001). Finally, reduced mean aortic AAA diameter was observed in AngII-infused mice treated with ApoA-I mimetic peptide compared with saline-injected controls. In conclusion, ApoA-I/HDL-C systemic levels are negatively associated with AAA evolution. Therapies targeting HDL functionality could halt AAA formation.

Purpose: To test whether red blood cell (RBC) membrane composition is modified in abdominal aortic aneurysms (AAA) patients., Experimental Design: RBC membrane extracts from AAA patients (aortic diameter >3 cm, n = 7) and control subjects (n = 4) were analyzed by a label-free quantitative MS-based strategy, using spectral count data. Additional validation was performed by western-blot., Results: Data analysis based on spectral count from MS/MS-based experiments provided us a signature of 39 proteins differentially expressed in RBC membranes between AAA and controls (changes equal/over 1.515-fold; p-values equal/lower 0.05). MS data revealed altered levels of structural membrane proteins (e.g. spectrins and ankyrin), components of the degradation machinery (proteasome subunits), and oxidative stress related proteins (e.g. catalase and peroxiredoxin-2) among others. Decreased catalase and peroxiredoxin-2 expression in RBC membrane of AAA patients compared to controls were further validated by Western blot, confirming the proteomic results., Conclusions and Clinical Relevance: RBCs membrane protein composition is altered in AAA patients, which could be involved in the pathological role of RBCs in aortic tissue and become potential targets to prevent AAA progression., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To find potential biomarkers of abdominal aortic aneurysms (AAA), we performed a differential proteomic study based on human plasma-derived microvesicles., Experimental Design: Exosomes and microparticles isolated from plasma of AAA patients and control subjects (n = 10 each group) were analyzed by a label-free quantitative MS-based strategy. Homemade and publicly available software packages have been used for MS data analysis., Results: The application of two kinds of bioinformatic tools allowed us to find differential protein profiles from AAA patients. Some of these proteins found by the two analysis methods belong to main pathological mechanisms of AAA such as oxidative stress, immune-inflammation, and thrombosis., Conclusions and Clinical Relevance: Data analysis from label-free MS-based experiments requires the use of sophisticated bioinformatic approaches to perform quantitative studies from complex protein mixtures. The application of two of these bioinformatic tools provided us a preliminary list of differential proteins found in plasma-derived microvesicles not previously associated to AAA, which could help us to understand the pathological mechanisms related to this disease., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Iron deposits are observed in tissue of abdominal aortic aneurysm (AAA) patients, although the underlying mechanisms are not completely elucidated. Therefore we explored circulating markers of iron metabolism in AAA patients, and tested if they could serve as biomarkers of AAA. Increased red blood cell (RBC)-borne iron retention and transferrin, transferrin receptor and ferritin expression was observed in AAA tissue compared to control aorta (immunohistochemistry and western blot). In contrast, decreased circulating iron, transferrin, mean corpuscular haemoglobin concentration (MCHC) and haemoglobin concentration, along with circulating RBC count, were observed in AAA patients (aortic diameter >3 cm, n=114) compared to controls (aortic diameter <3 cm, n=88) (ELISA), whereas hepcidin concentrations were increased in AAA subjects (MS/MS assay). Moreover, iron, transferrin and haemoglobin levels were negatively, and hepcidin positively, correlated with aortic diameter in AAA patients. The association of low haemoglobin with AAA presence or aortic diameter was independent of specific risk factors. Moreover, MCHC negatively correlated with thrombus area in another cohort of AAA patients (aortic diameter 3-5 cm, n=357). We found that anaemia was significantly more prevalent in AAA patients (aortic diameter >5 cm, n=8,912) compared to those in patients with atherosclerotic aorto-iliac occlusive disease (n=17,737) [adjusted odds ratio=1.77 (95% confidence interval: 1.61;1.93)]. Finally, the mortality risk among AAA patients with anaemia was increased by almost 30% [adjusted hazard ratio: 1.29 (95% confidence interval: 1.16;1.44)] as compared to AAA subjects without anaemia. In conclusion, local iron retention and altered iron recycling associated to high hepcidin and low transferrin systemic concentrations could lead to reduced circulating haemoglobin levels in AAA patients. Low haemoglobin levels are independently associated to AAA presence and clinical outcome.

Objective: To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA)., Approach and Results: Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry-based strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity software highlighted the presence of several circulating proteins, among them were proteins from the complement system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA diameter=3-5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally, the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen species production., Conclusions: A decrease of systemic C3 concentration and activity in the later stages of AAA associated with local complement retention, consumption, and proteolysis in the thrombus could induce PMN chemotaxis and activation, playing a detrimental role in AAA progression.

In the search for novel biomarkers, noncandidate-based proteomic strategies open up new opportunities to gain a deeper insight into disease processes regarding their molecular mechanisms, the risk factors involved, and the monitoring of disease progression. To carry out these complex analyses, the combined use of gel electrophoresis with mass spectrometry (MS) represents a powerful choice. In addition, the introduction of protein dye labeling has notably improved the reliability of differential expression studies by increasing the statistical significance of the protein candidates. Here, we describe a strategy where different layers (luminal/abluminal) from the intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) patients were incubated in protein-free medium. Then, the levels of the proteins released were compared by two-dimensional differential in-gel electrophoresis (2D-DIGE) and the proteins of interest identified by MS. We consider that the use of tissue-conditioned media could offer a substantial advantage in the analytical study of biological fluids, as they provide a source of proteins to be released to the bloodstream, which could serve as potential circulating biomarkers.

Oxidative stress is involved in the chronic pathological vascular remodelling of both abdominal aortic aneurysm and occlusive atherosclerosis. Red blood cells (RBCs), leukocytes and platelets present in both, aneurysmal intraluminal thrombus and intraplaque haemorraghes, could be involved in the redox imbalance inside diseased arterial tissues. RBCs haemolysis may release the pro-oxidant haemoglobin (Hb), which transfers heme to tissue and low-density lipoproteins. Heme-iron potentiates molecular, cell and tissue toxicity mediated by leukocytes and other sources of reactive oxygen species (ROS). Polymorphonuclear neutrophils release myeloperoxidase and, along with activated platelets, produce superoxide mediated by NADPH oxidase, causing oxidative damage. In response to this pro-oxidant milieu, several antioxidant molecules of plasma or cell origin can prevent ROS production. Free Hb binds to haptoglobin (Hp) and once Hp-Hb complex is endocytosed by CD163, liberated heme is converted into less toxic compounds by heme oxygenase-1. Iron homeostasis is mainly regulated by transferrin, which transports ferric ions to other cells. Transferrin-bound iron is internalised via endocytosis mediated by transferrin receptor. Once inside the cell, iron is mainly stored by ferritin. Other non hemo-iron related antioxidant enzymes (e.g. superoxide dismutase, catalase, thioredoxin and peroxiredoxin) are also involved in redox modulation in vascular remodelling. Oxidative stress is a main determinant of chronic pathological remodelling of the arterial wall, partially linked to the presence of RBCs, leukocytes, platelets and oxidised fibrin within tissue and to the imbalance between pro-/anti-oxidant molecules. Understanding the complex mechanisms underlying redox imbalance could help to define novel potential targets to decrease atherothrombotic risk.

Abdominal aortic aneurysm (AAA) is an important health problem, both because of AAA rupture and death and because of increased cardiovascular mortality. Identification of new biomarkers of AAA may suggest novel pathological mechanisms and targets for new medical treatments to slow AAA progression. Metabolic changes in AAA patients were mainly related to carbohydrate and lipid metabolism and many of these changes can be associated with a situation of insulin resistance (which can be related to metabolic syndrome) together with altered amino acid metabolism. For the first time, metabolites that can be associated with differential metabolism by the gut microflora of AAA patients have also been found. Moreover, aminomalonic acid in plasma has been shown to be the metabolite with the biggest difference between patients suffering from large aneurysm (>5 cm) and controls.

Cell stress proteins (CSPS) are a large and heterogeneous family of proteins, sharing two main characteristics: their levels and/or location are modified under stress and most of them can exert a chaperon function inside the cells. Nonetheless, they are also involved in the modulation of several mechanisms, both at the intracellular and the extracellular compartments. There are more than 100 proteins belonging to the CSPs family, among them the thioredoxin (TRX) system, which is the focus of the present paper. TRX system is composed of several proteins such as TRX and peroxiredoxin (PRDX), two thiol-containing enzymes that are key players in redox homeostasis due to their ability to scavenge potential harmful reactive oxygen species. In addition to their main role as antioxidants, recent data highlights their function in several processes such as cell signalling, immune inflammatory responses, or apoptosis, all of them key mechanisms involved in atherothrombosis. Moreover, since TRX and PRDX are present in the pathological vascular wall and can be secreted under prooxidative conditions to the circulation, several studies have addressed their role as diagnostic, prognostic, and therapeutic biomarkers of cardiovascular diseases (CVDs).

Objective: Polymorphonuclear neutrophils (PMNs) play a main role in abdominal aortic aneurysm (AAA) progression. We have analyzed circulating PMNs isolated from AAA patients and controls by a proteomic approach to identify proteins potentially involved in AAA pathogenesis., Methods and Results: PMNs from 8 AAA patients (4 large AAA >5 cm and 4 small AAA 3-5 cm) and 4 controls were analyzed by 2D differential in-gel electrophoresis. Among differentially expressed spots, several proteins involved in redox balance were identified by mass spectrometry (eg, cyclophilin, thioredoxin reductase, catalase). Diminished catalase expression and activity were observed in PMNs from AAA patients compared with controls. In contrast, PMNs from AAA patients displayed higher H(2)O(2) and myeloperoxidase levels than PMNs from controls. Moreover, a significant decrease in catalase mRNA levels was observed in PMNs after phorbol 12-myristate 13-acetate incubation. Catalase plasma levels were also decreased in large (n=47) and small (n=56) AAA patients compared with controls (n=34). We observed catalase expression in AAA thrombus and thrombus-conditioned medium, associated with PMN infiltration. Furthermore, increased H(2)O(2) levels were observed in AAA thrombus-conditioned medium compared with the media layer., Conclusions: Diminished catalase levels in circulating PMNs and plasma are observed in AAA patients, supporting an important role of oxidative stress in AAA evolution.

Objective: In the search of novel biomarkers of abdominal aortic aneurysm (AAA) progression, proteins released by intraluminal thrombus (ILT) were analyzed by a differential proteomic approach., Methods and Results: Different layers (luminal/abluminal) of the ILT of AAA were incubated, and the proteins released were analyzed by 2-dimensional difference in-gel electrophoresis. Several differentially expressed proteins involved in main AAA pathological mechanisms (proteolysis, oxidative stress, and thrombosis) were identified by mass spectrometry. Among the proteins identified, peroxiredoxin-1 (PRX-1) was more released by the luminal layer compared with the abluminal layer of the ILT, which was further validated by Western blot, ELISA, and immunohistochemistry. We demonstrated increased PRX-1 serum levels in AAA patients compared with healthy subjects and also positive correlation among PRX-1 and AAA diameter, plasmin-antiplasmin, and myeloperoxidase levels. Finally, a prospective study revealed a positive correlation between PRX-1 serum levels and AAA expansion rate. Moreover, the combination of PRX-1 and AAA size had significantly additive value in predicting growth., Conclusions: Several proteins associated with AAA pathogenesis have been identified by a proteomic approach in ILT-conditioned medium. Among them, PRX-1 serum levels are increased in AAA patients and correlate with AAA size and growth rate, suggesting the potential use of PRX-1 as a biomarker for AAA evolution.

Atherothrombosis remains a major cause of morbidity and mortality in the western world. The underlying processes associated with clinical expression of atherothrombosis include oxidative stress and proteolysis in relation to neovascularisation and intraplaque hemorrhages, leading to immuno-inflammatory response, cell death, and extracellular matrix breakdown. The complex biological multifactorial nature of atherothrombosis requires the development of novel technologies that allow the analysis of cellular and molecular processes responsible for the transition to disease phenotypes and the discovery of new diagnostic and prognostic biomarkers. In the present article, we have reviewed recent advances in the application of proteomic and metabolomic techniques to the study of atherothrombosis. We have focused on recent studies analyzing cells involved in hemo-thrombus formation (platelets, red blood cells, and polymorphonuclear cells), as well as tissues, tissue-conditioned media, and plasma of atherothrombotic patients. In the future, the application of these high-throughput technologies, along with imaging techniques, in systems biology approaches will help to individualize medicine.

The advent of metal cluster as a primary ion source in the late 1980s, made it feasible to probe surfaces for complex organic structures due to a reduced in-source fragmentation, and opened the door to the direct analysis of biological samples. Despite the mass range measurable by TOF-secondary ion MS (SIMS) still being rather limited, the information obtained from cells and tissues comes together with the technical innovations introduced in the last decade. In this article, we give a brief overview of the technique itself and make some emphasis on the advances in the last three years in the analysis of biological surfaces, particularly those with direct implication in the biomedical field; reviewing what kind of information this instrumentation will add to current tool in pathology.

Atherothrombosis is the underlying cause of several clinical manifestations, such as acute coronary syndromes, cerebrovascular disease, and peripheral artery disease, which together are the leading cause of death in the Western world. Proteins from vascular cells or atherosclerotic plaques that are present in plasma are modified along the different steps of atherosclerotic development and constitute target candidates for vascular research, particularly in the search for novel biological markers of cardiovascular risk. In this review, we summarize proteomic techniques and the most recent results obtained by application of these high-throughput strategies to cardiovascular samples.