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While COVID-19’s urgency has diminished since its emergence in late 2019, it remains a significant public health challenge. Recent research reveals that the molecular intricacies of this virus are far more complex than initially understood, with numerous post-translational modifications leading to diverse proteoforms and viral particle heterogeneity. Mass spectrometry-based proteomics of patient serum/plasma emerges as a promising complementary approach to traditional diagnostic methods, offering insights into SARS-CoV-2 protein dynamics and enhancing understanding of the disease and its long-term consequences. This article highlights key findings from three years of pandemic-era proteomics research. It delves into biomarker discovery, diagnostic advancements, and drug development efforts aimed at monitoring COVID-19 onset and progression and exploring treatment options. Additionally, it examines global protein abundance and post-translational modification profiling to elucidate signaling pathway alterations and protein-protein interactions during infection. Finally, it explores the potential of emerging multi-omics analytic strategies in combatting SARS-CoV-2.

Neutrophils play a pathogenic role in COVID-19 by releasing Neutrophils Extracellular Traps (NETs) or human neutrophil elastase (HNE). Given that HNE is inhibited by α1-antitrypsin (AAT), we aimed to assess the content of HNE, α1-antitrypsin (AAT) and HNE–AAT complexes (the AAT/HNE balance) in 33 bronchoalveolar lavage fluid (BALf) samples from COVID-19 patients. These samples were submitted for Gel-Electrophoresis, Western Blot and ELISA, and proteins (bound to AAT or HNE) were identified by Liquid Chromatography-Mass Spectrometry. NETs’ release was analyzed by confocal microscopy. Both HNE and AAT were clearly detectable in BALf at high levels. Contrary to what was previously observed in other settings, the formation of HNE–AAT complex was not detected in COVID-19. Rather, HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free AAT to bind to histones. While confirming this binding, AAT was not able to inhibit NET formation. In conclusion, despite the finding of a high burden of free and bound HNE, the lack of the HNE–AAT inhibitory complex in COVID-19 BALf demonstrates that AAT is not able to block HNE activity. Furthermore, while binding to histones, AAT does not prevent NET formation nor their noxious activity.

Human neutrophil elastase (HNE) is involved in SARS-CoV-2 virulence and plays a pivotal role in lung infection of patients infected by COVID-19. In healthy individuals, HNE activity is balanced by α1-antitrypsin (AAT). This is a 52 kDa glycoprotein, mainly produced and secreted by hepatocytes, that specifically inhibits HNE by blocking its activity through the formation of a stable complex (HNE–AAT) in which the two proteins are covalently bound. The lack of this complex, together with the detection of HNE activity in BALf/plasma samples of COVID-19 patients, leads us to hypothesize that potential functional deficiencies should necessarily be attributed to possible structural modifications of AAT. These could greatly diminish its ability to inhibit neutrophil elastase, thus reducing lung protection. The aim of this work was to explore the oxidation state of AAT in BALf/plasma samples from these patients so as to understand whether the deficient inhibitory activity of AAT was somehow related to possible conformational changes caused by the presence of abnormally oxidized residues.

In Antarctica, ice-free areas can be found along the coast, on mountain peaks, and in the McMurdo Dry Valleys, where microorganisms well-adapted to harsh conditions can survive and reproduce. Metabolic analyses can shed light on the survival mechanisms of Antarctic soil communities from both coastal sites, under different plant coverage stages, and inner sites where slow-growing or dormant microorganisms, low water availability, salt accumulation, and a limited number of primary producers make metabolomic profiling difficult. Here, we report, for the first time, an efficient protocol for the extraction and the metabolic profiling of Antarctic soils based on the combination of NMR spectroscopy and mass spectrometry (MS). This approach was set up on samples harvested along different localities of Victoria Land, in continental Antarctica, devoid of or covered by differently developed biological crusts. NMR allowed for the identification of thirty metabolites (mainly sugars, amino acids, and organic acids) and the quantification of just over twenty of them. UPLC-MS analysis identified more than twenty other metabolites, in particular flavonoids, medium- and long-chain fatty acids, benzoic acid derivatives, anthracenes, and quinones. Our results highlighted the complementarity of the two analytical techniques. Moreover, we demonstrated that their combined use represents the “gold standard” for the qualitative and quantitative analysis of little-explored samples, such as those collected from Antarctic soils.

The Burkholderia cepacia complex comprises environmental and clinical Gram-negative bacteria that infect particularly debilitated people, such as those with cystic fibrosis. Their high level of antibiotic resistance makes empirical treatments often ineffective, increasing the risk of worst outcomes and the diffusion of multi-drug resistance. However, the discovery of new antibiotics is not trivial, so an alternative can be the use of vaccination. Here, the reverse vaccinology approach has been used to identify antigen candidates, obtaining a short-list of 24 proteins. The localization and different aspects of virulence were investigated for three of them—BCAL1524, BCAM0949, and BCAS0335. The three antigens were localized in the outer membrane vesicles confirming that they are surface exposed. We showed that BCAL1524, a collagen-like protein, promotes bacteria auto-aggregation and plays an important role in virulence, in the Galleria mellonella model. BCAM0949, an extracellular lipase, mediates piperacillin resistance, biofilm formation in Luria Bertani and artificial sputum medium, rhamnolipid production, and swimming motility; its predicted lipolytic activity was also experimentally confirmed. BCAS0335, a trimeric adhesin, promotes minocycline resistance, biofilm organization in LB, and virulence in G. mellonella. Their important role in virulence necessitates further investigations to shed light on the usefulness of these proteins as antigen candidates.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. Methods Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. Results ICU patients showed a marked increase in neutrophils (1.24 × 105 ml− 1, 0.85–2.07), lower lymphocyte (0.97 × 105 ml− 1, 0.024–0.34) and macrophages fractions (0.43 × 105 ml− 1, 0.34–1.62) compared to IMW patients (0.095 × 105 ml− 1, 0.05–0.73; 0.47 × 105 ml− 1, 0.28–1.01 and 2.14 × 105 ml− 1, 1.17–3.01, respectively) (p

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-β, IL8 and IL1β). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.

(1) Background: Chronic Kidney Disease (CKD) induces metabolic derangement of amino acid (AA) kinetics, eliciting severe damage to the protein anabolism. This damage is further intensified by a significant loss of AAs through hemodialysis (HD), affecting all tissues with a high metabolic turnover, such as the myocardium and body muscle mass. (2) Aim: to illustrate the effects of a novel AA mixture in boosting mitochondrial energy production. (3) Methods: A strict selection of 164 dialysis patients was carried out, allowing us to finally identify 22 compliant patients who had not used any form of supplements over the previous year. The study design envisaged a 6-month randomized, double-blind trial for the comparison of two groups of hemodialysis patients: eleven patients (67.2 ± 9.5 years) received the novel AA mix (TRG), whilst the other eleven (68.2 ± 10.5 years) were given a placebo mix that was indistinguishable from the treatment mix (PLG). (4) Results: Despite the 6-month observation period, the following were observed: maintenance of target hemoglobin values with a reduced need for erythropoiesis-stimulating agents in TRG > 36% compared to PLG (p < 0.02), improved phase angle (PhA) accompanied by an increase in muscle mass solely in the TRG group (p < 0.05), improved Left Ventricular Ejection Fraction (LVEF > 67%) in the TRG versus PLG group (p < 0.05) with early but marked signs of improved diastolic function. Increased sensitivity to insulin with greater control of glycemic levels in TRG versus PLG (p = 0.016). (5) Conclusions: the new AA mix seemed to be effective, showing a positive result on nutritional metabolism and cardiac performance, stable hemoglobin levels with the need for lower doses of erythropoietin (EPO), insulin increased cell sensitivity, better muscle metabolism with less loss of mass.

The identification of markers of inflammatory activity at the early stages of pulmonary diseases which share common characteristics that prevent their clear differentiation is of great significance to avoid misdiagnosis, and to understand the intrinsic molecular mechanism of the disorder. The combination of electrophoretic/chromatographic methods with mass spectrometry is currently a promising approach for the identification of candidate biomarkers of a disease. Since the fluid phase of sputum is a rich source of proteins which could provide an early diagnosis of specific lung disorders, it is frequently used in these studies. This report focuses on the state-of-the-art of the application, over the last ten years (2011–2021), of sputum proteomics in the investigation of severe lung disorders such as COPD; asthma; cystic fibrosis; lung cancer and those caused by COVID-19 infection. Analysis of the complete set of proteins found in sputum of patients affected by these disorders has allowed the identification of proteins whose levels change in response to the organism’s condition. Understanding proteome dynamism may help in associating these proteins with alterations in the physiology or progression of diseases investigated.

Nasu-Hakola Disease (NHD) is a recessively inherited systemic leukodystrophy disorder characterized by a combination of frontotemporal presenile dementia and lytic bone lesions. NHD is known to be genetically related to a structural defect of TREM2 and DAP12, two genes that encode for different subunits of the membrane receptor signaling complex expressed by microglia and osteoclast cells. Because of its rarity, molecular or proteomic studies on this disorder are absent or scarce, only case reports based on neuropsychological and genetic tests being reported. In light of this, the aim of this paper is to provide evidence on the potential of a label-free proteomic platform based on the Multidimensional Protein Identification Technology (MudPIT), combined with in-house software and on-line bioinformatics tools, to characterize the protein expression trends and the most involved pathways in NHD. The application of this approach on the Lymphoblastoid cells from a family composed of individuals affected by NHD, healthy carriers and control subjects allowed for the identification of about 3000 distinct proteins within the three analyzed groups, among which proteins anomalous to each category were identified. Of note, several differentially expressed proteins were associated with neurodegenerative processes. Moreover, the protein networks highlighted some molecular pathways that may be involved in the onset or progression of this rare frontotemporal disorder. Therefore, this fully automated MudPIT platform which allowed, for the first time, the generation of the whole protein profile of Lymphoblastoid cells from Nasu-Hakola subjects, could be a valid approach for the investigation of similar neurodegenerative diseases.

The aim of the present report is to review the literature addressing the methods developed for the purification of alpha1-antitrypsin (AAT) from the 1950s to the present. AAT is a glycoprotein whose main function is to protect tissues from human neutrophil elastase (HNE) and other proteases released by neutrophils during an inflammatory state. The lack of this inhibitor in human serum is responsible for the onset of alpha1-antitrypsin deficiency (AATD), which is a severe genetic disorder that affects lungs in adults and for which there is currently no cure. Being used, under special circumstances, as a medical treatment of AATD in the so-called “replacement” therapy (consisting in the intravenous infusion of the missing protein), AAT is a molecule with a lot of therapeutic importance. For this reason, interest in AAT purification from human plasma or its production in a recombinant version has grown considerably in recent years. This article retraces all technological advances that allowed the manufacturers to move from a few micrograms of partially purified AAT to several grams of highly purified protein. Moreover, the chronic augmentation and maintenance therapy in individuals with emphysema due to congenital AAT deficiency (current applications in the clinical setting) is also presented.

Collagen Tissue Disease−associated Interstitial Lung Fibrosis (CTD-ILDs) and Bronchiolitis Obliterans Syndrome (BOS) represent severe lung fibrogenic disorders, characterized by fibro-proliferation with uncontrolled extracellular matrix deposition. Hyaluronic acid (HA) plays a key role in fibrosis with its specific receptor, CD44, overexpressed by CTD-ILD and BOS cells. The aim is to use HA-liposomes to develop an inhalatory treatment for these diseases. Liposomes with HA of two molecular weights were prepared and characterized. Targeting efficiency was assessed toward CTD-ILD and BOS cells by flow cytometry and confocal microscopy and immune modulation by RT-PCR and ELISA techniques. HA-liposomes were internalized by CTD-ILD and BOS cells expressing CD44, and this effect increased with higher HA MW. In THP-1 cells, HA-liposomes decreased pro-inflammatory cytokines IL-1β, IL-12, and anti-fibrotic VEGF transcripts but increased TGF-β mRNA. However, upon analyzing TGF-β release from healthy donors-derived monocytes, we found liposomes did not alter the release of active pro-fibrotic cytokine. All liposomes induced mild activation of neutrophils regardless of the presence of HA. HA liposomes could be also applied for lung fibrotic diseases, being endowed with low pro-inflammatory activity, and results confirmed that higher MW HA are associated to an increased targeting efficiency for CD44 expressing LFs-derived from BOS and CTD-ILD patients.