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The palladium-catalyzed reaction of N-protected 2-indolylmethyl acetates with soft carbon pronucleophiles is described. Besides the formation of the expected coupling reaction at the C1' position, unprecedented attack at the C3 position of the plausible η3-indolyl-palladium intermediate has been observed, and the selectivity control C1'/C3 seems to depend on the nature of the protecting group and ligand. The reactivity of 3-indolylmethyl acetates has also been also investigated. Quantum chemical calculations support the experimental results.
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This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin ß4 and thymosin ß10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin ß4 and thymosin ß10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of ß-thymosins in health and disease. The functions of ß-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "ß-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development.
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Timosina , Humanos , Timosina/metabolismo , Timosina/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Mitochondrial diseases (MDs) affect 4300 individuals, with different ages of presentation and manifestation in any organ. How defects in mitochondria can cause such a diverse range of human diseases remains poorly understood. In recent years, several published research articles regarding the metabolic and protein profiles of these neurogenetic disorders have helped shed light on the pathogenetic mechanisms. By investigating different pathways in MDs, often with the aim of identifying disease biomarkers, it is possible to identify molecular processes underlying the disease. In this perspective, omics technologies such as proteomics and metabolomics considered in this review, can support unresolved mitochondrial questions, helping to improve outcomes for patients.
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Biomarcadores , Metabolómica , Mitocondrias , Enfermedades Mitocondriales , Proteómica , Humanos , Metabolómica/métodos , Mitocondrias/metabolismo , Proteómica/métodos , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/diagnóstico , AnimalesRESUMEN
Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin ß4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of α-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. α-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin ß4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , alfa-Defensinas , Humanos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Cistatina B/análisis , Cistatina B/metabolismo , Proteómica/métodos , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/metabolismo , Enfermedades Neurodegenerativas/metabolismo , alfa-Defensinas/análisis , alfa-Defensinas/metabolismo , Saliva/química , Proteínas y Péptidos Salivales/metabolismo , Factores de Transcripción/metabolismo , Biomarcadores/análisisRESUMEN
Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.
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Seminoma , Neoplasias Testiculares , Humanos , Masculino , Línea Celular Tumoral , Citoplasma/metabolismo , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 2 de la Matriz/metabolismo , Proteómica , Securina/genética , Securina/metabolismo , Seminoma/genética , Espectrina/genética , Neoplasias Testiculares/genéticaRESUMEN
Recently, advances in molecular biology and bioinformatics have allowed a more thorough understanding of tumorigenesis in aggressive PitNETs (pituitary neuroendocrine tumors) through the identification of specific essential genes, crucial molecular pathways, regulators, and effects of the tumoral microenvironment. Target therapies have been developed to cure oncology patients refractory to traditional treatments, introducing the concept of precision medicine. Preliminary data on PitNETs are derived from preclinical studies conducted on cell cultures, animal models, and a few case reports or small case series. This study comprehensively reviews the principal pathways involved in aggressive PitNETs, describing the potential target therapies. A search was conducted on Pubmed, Scopus, and Web of Science for English papers published between 1 January 2004, and 15 June 2023. 254 were selected, and the topics related to aggressive PitNETs were recorded and discussed in detail: epigenetic aspects, membrane proteins and receptors, metalloprotease, molecular pathways, PPRK, and the immune microenvironment. A comprehensive comprehension of the molecular mechanisms linked to PitNETs' aggressiveness and invasiveness is crucial. Despite promising preliminary findings, additional research and clinical trials are necessary to confirm the indications and effectiveness of target therapies for PitNETs.
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Tumores Neuroendocrinos , Neoplasias Hipofisarias , Animales , Humanos , Neoplasias Hipofisarias/patología , Hipófisis/metabolismo , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/terapia , Tumores Neuroendocrinos/metabolismo , Agresión , Microambiente Tumoral/genéticaRESUMEN
In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.
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Proteoma , Proteínas y Péptidos Salivales , Humanos , Procesamiento Proteico-Postraduccional , Glicosilación , ProteolisisRESUMEN
Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases that target the liver and have a wide spectrum of presentation. A global overview of quantitative variations on the salivary proteome in presence of these two pathologies is investigated in this study. The acid-insoluble salivary fraction of AIH and PBC patients, and healthy controls (HCs), was analyzed using a gel-based bottom-up proteomic approach combined with a robust machine learning statistical analysis of the dataset. The abundance of Arginase, Junction plakoglobin, Desmoplakin, Hexokinase-3 and Desmocollin-1 decreased, while that of BPI fold-containing family A member 2 increased in AIHp compared to HCs; the abundance of Gelsolin, CD14, Tumor-associated calcium signal transducer 2, Clusterin, Heterogeneous nuclear ribonucleoproteins A2/B1, Cofilin-1 and BPI fold-containing family B member 2 increased in PBCp compared to HCs. The abundance of Hornerin decreased in both AIHp and PBCp with respect to HCs and provided an area under the ROC curve of 0.939. Machine learning analysis confirmed the feasibility of the salivary proteome to discriminate groups of subjects based on AIH or PBC occurrence as previously suggested by our group. The topology-based functional enrichment analysis performed on these potential salivary biomarkers highlights an enrichment of terms mostly related to the immune system, but also with a strong involvement in liver fibrosis process and with antimicrobial activity.
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Enfermedades Autoinmunes , Hepatitis Autoinmune , Cirrosis Hepática Biliar , Hepatopatías , Humanos , Proteoma , ProteómicaRESUMEN
The synthesis of 2,3-dihydropyrazino[1,2-a]indol-4(1H)-ones from the sequential reaction of amino acid methyl esters with readily available indole-2-ylmethyl acetates is described. The reaction proceeds in situ under basic conditions of highly unstable and reactive 2-alkylideneindolenines followed by Michael-type addition of α-amino acid methyl esters/intramolecular cyclization.
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Cystatin B is a small, multifunctional protein involved in the regulation of inflammation, innate immune response, and neuronal protection and found highly abundant in the brains of patients with Alzheimer's disease (AD). Recently, our study demonstrated a significant association between the level of salivary cystatin B and AD. Since the protein is able to establish protein-protein interaction (PPI) in different contexts and aggregation-prone proteins and the PPI networks are relevant for AD pathogenesis, and due to the relevance of finding new AD markers in peripheral biofluids, we thought it was interesting to study the possible involvement of cystatin B in PPIs in saliva and to evaluate differences and similarities between AD and age-matched elderly healthy controls (HC). For this purpose, we applied a co-immunoprecipitation procedure and a bottom-up proteomics analysis to purify, identify, and quantify cystatin B interactors. Results demonstrated for the first time the existence of a salivary cystatin B-linked multi-protein complex composed by 82 interactors and largely expressed in the body. Interactors are involved in neutrophil activation, antimicrobial activity, modulation of the cytoskeleton and extra-cellular matrix (ECM), and glucose metabolism. Preliminary quantitative data showed significantly lower levels of triosophosphate isomerase 1 and higher levels of mucin 7, BPI, and matrix Gla protein in AD with respect to HC, suggesting implications associated with AD of altered glucose metabolism, antibacterial activities, and calcification-associated processes. Data are available via ProteomeXchange with identifiers PXD039286 and PXD030679.
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INTRODUCTION: Arginine metabolism is involved in the regulation of several biological processes. Many liquid chromatography tandem-mass spectrometry methods for the determination of arginine and its metabolites have been developed but they are time consuming and imply long pre-analytical procedures. The purpose of this study was to develop a rapid method for the simultaneous determination of arginine, citrulline, ornithine, symmetric and asymmetric dimethylarginine and monomethylarginine in human plasma. MATERIALS AND METHODS: The pre-analytical procedure consisted in a simple deproteinization. The chromatographic separation was performed using hydrophilic interaction liquid chromatography. Analytes detection was performed with a triple quadrupole equipped with electrospray ion source operating in positive ion mode. Mass spectrometry experiments were conducted in multiple reaction monitoring mode. RESULTS AND CONCLUSIONS: Recovery ranged from 92.2 to 108.0%. The within-run imprecision and between-run imprecision ranged from 1.5 to 6.8 % and 3.8 to 11.9%, respectively. Carry over and matrix effect did not affect quantitative analysis. Extraction recovery was between 95 and 105 %. Stability after pre-analytical procedure was tested and all the metabolites were stable after 48 h at 4 °C. In conclusion, our novel method allow a rapid and easy determination of arginine and its metabolites both for research and clinical routine use.
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Arginina , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida/métodos , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los ResultadosRESUMEN
(1) Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases characterized by chronic hepatic inflammation and progressive liver fibrosis. The possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. The use of proteomics for personalized medicine is a rapidly emerging field. (2) Salivary proteomic data of 36 healthy controls (HCs), 36 AIH and 36 PBC patients, obtained by liquid chromatography/mass spectrometry top-down pipeline, were analyzed by multiple Mann-Whitney test, Kendall correlation, Random Forest (RF) analysis and Linear Discriminant Analysis (LDA); (3) Mann-Whitney tests provided indications on the panel of differentially expressed salivary proteins and peptides, namely cystatin A, statherin, histatin 3, histatin 5 and histatin 6, which were elevated in AIH patients with respect to both HCs and PBC patients, while S100A12, S100A9 short, cystatin S1, S2, SN and C showed varied levels in PBC with respect to HCs and/or AIH patients. RF analysis evidenced a panel of salivary proteins/peptides able to classify with good accuracy PBC vs. HCs (83.3%), AIH vs. HCs (79.9%) and PBC vs. AIH (80.2%); (4) RF appears to be an attractive machine-learning tool suited for classification of AIH and PBC based on their different salivary proteomic profiles.
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Enfermedades Autoinmunes , Hepatitis Autoinmune , Cirrosis Hepática Biliar , Hepatopatías , Humanos , Proteómica , Histatinas , Hepatopatías/diagnóstico , Enfermedades Autoinmunes/diagnóstico , Hepatitis Autoinmune/diagnóstico , Proteínas y Péptidos Salivales , BiomarcadoresRESUMEN
Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese.
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Queso , Microbiota , Salud Única , Animales , Queso/análisis , Cabras/genética , ARN Ribosómico 16S/genética , Bacterias/genética , Microbiota/genéticaRESUMEN
BACKGROUND: Aging is a risk factor for several pathologies as Alzheimer's disease (AD). Great interest exists, therefore, in discovering diagnostic biomarkers and indicators discriminating biological aging and health status. To this aim, omic investigations of biological matrices, as saliva, whose sampling is easy and non-invasive, offer great potential. OBJECTIVE: Investigate the salivary proteome through a statistical comparison of the proteomic data by several approaches to highlight quali-/quantitative variations associated specifically either to aging or to AD occurrence, and, thus, able to classify the subjects. METHODS: Salivary proteomic data of healthy controls under-70 (adults) and over-70 (elderly) years old, and over-70 AD patients, obtained by liquid chromatography/mass spectrometry, were analyzed by multiple Mann-Whitney test, Kendall correlation, and Random-Forest (RF) analysis. RESULTS: Almost all the investigated proteins/peptides significantly decreased in relation to aging in elderly subjects, with or without AD, in comparison with adults. AD subjects exhibited the highest levels of α-defensins, thymosin ß4, cystatin B, S100A8 and A9. Correlation tests also highlighted age/disease associated differences. RF analysis individuated quali-/quantitative variations in 20 components, as oxidized S100A8 and S100A9, α-defensin 3, P-B peptide, able to classify with great accuracy the subjects into the three groups. CONCLUSION: The findings demonstrated a strong change of the salivary protein profile in relation to the aging. Potential biomarkers candidates of AD were individuated in peptides/proteins involved in antimicrobial defense, innate immune system, inflammation, and in oxidative stress. RF analysis revealed the feasibility of the salivary proteome to discriminate groups of subjects based on age and health status.
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Enfermedad de Alzheimer , alfa-Defensinas , Anciano , Envejecimiento , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/metabolismo , Calgranulina A , Cistatina B/metabolismo , Humanos , Proteoma/metabolismo , Proteómica/métodos , Proteínas y Péptidos Salivales/metabolismo , alfa-Defensinas/metabolismoRESUMEN
The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role of the correct disulfide in the attainment of the native structure. CD analysis, mass spectrometry, fluorescence spectroscopy and enzyme activity indicate that native disulfides drive the formation of the secondary and tertiary structures that cannot be entirely formed in their absence. This opposes a common opinion that these structures are first formed and then stabilized by the native disulfides. Our results also indicate that a spontaneous re-oxidation of a reduced RNase cannot produce a complete recovery of activity, as described by many textbooks; this can be obtained only in the presence of a reshuffling solution such as GSH/GSSG.
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Disulfuros , Pliegue de Proteína , Disulfuros/química , Oxidación-Reducción , Ribonucleasa Pancreática/metabolismo , RibonucleasasRESUMEN
The present investigation aimed to explore the intact proteome of tissues of pediatric brain tumors of different WHO grades and localizations, including medulloblastoma, pilocytic astrocytoma, and glioblastoma, in comparison with the available data on ependymoma, to contribute to the understanding of the molecular mechanisms underlying the onset and progression of these pathologies. Tissues have been homogenized in acidic water−acetonitrile solutions containing proteases inhibitors and analyzed by LC−high resolution MS for proteomic characterization and label-free relative quantitation. Tandem MS spectra have been analyzed by either manual inspection or software elaboration, followed by experimental/theoretical MS fragmentation data comparison by bioinformatic tools. Statistically significant differences in protein/peptide levels between the different tumor histotypes have been evaluated by ANOVA test and Tukey's post-hoc test, considering a p-value > 0.05 as significant. Together with intact protein and peptide chains, in the range of molecular mass of 1.3−22.8 kDa, several naturally occurring fragments from major proteins, peptides, and proteoforms have been also identified, some exhibiting proper biological activities. Protein and peptide sequencing allowed for the identification of different post-translational modifications, with acetylations, oxidations, citrullinations, deamidations, and C-terminal truncations being the most frequently characterized. C-terminal truncations, lacking from two to four amino acid residues, particularly characterizing the ß-thymosin peptides and ubiquitin, showed a different modulation in the diverse tumors studied. With respect to the other tumors, medulloblastoma, the most frequent malignant brain tumor of the pediatric age, was characterized by higher levels of thymosin ß4 and ß10 peptides, the latter and its des-IS form particularly marking this histotype. The distribution pattern of the C-terminal truncated forms was also different in glioblastoma, particularly underlying gender differences, according to the definition of male and female glioblastoma as biologically distinct diseases. Glioblastoma was also distinguished for the peculiar identification of the truncated form of the α-hemoglobin chain, lacking the C-terminal arginine, and exhibiting oxygen-binding and vasoconstrictive properties different from the intact form. The proteomic characterization of the undigested proteome, following the top-down approach, was challenging to originally investigate the post-translational events that differently characterize pediatric brain tumors. This study provides a contribution to elucidate the molecular profiles of the solid tumors most frequently affecting the pediatric age, and which are characterized by different grades of aggressiveness and localization.
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Neoplasias Encefálicas , Neoplasias Cerebelosas , Glioblastoma , Meduloblastoma , Neoplasias Encefálicas/metabolismo , Niño , Femenino , Humanos , Masculino , Péptidos/química , Proteoma/metabolismo , Proteómica/métodosRESUMEN
[This corrects the article DOI: 10.3389/fnins.2021.668852.].
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Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin ß4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538.
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Basic and preclinical research founded the progress of personalized medicine by providing a prodigious amount of integrated profiling data and by enabling the development of biomedical applications to be implemented in patient-centered care and cures. If the rapid development of genomics research boosted the birth of personalized medicine, further development in omics technologies has more recently improved our understanding of the functional genome and its relevance in profiling patients' phenotypes and disorders. Concurrently, the rapid biotechnological advancement in diverse research areas enabled uncovering disease mechanisms and prompted the design of innovative biological treatments tailored to individual patient genotypes and phenotypes. Research in stem cells enabled clarifying their role in tissue degeneration and disease pathogenesis while providing novel tools toward the development of personalized regenerative medicine strategies. Meanwhile, the evolving field of integrated omics technologies ensured translating structural genomics information into actionable knowledge to trace detailed patients' molecular signatures. Finally, neuroscience research provided invaluable models to identify preclinical stages of brain diseases. This review aims at discussing relevant milestones in the scientific progress of basic and preclinical research areas that have considerably contributed to the personalized medicine revolution by bridging the bench-to-bed gap, focusing on stem cells, omics technologies, and neuroscience fields as paradigms.
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The study investigates the role of the oxidative and proteolytic inactivation of alpha-1 antitrypsin (AAT) in the pathogenesis of bronchopulmonary dysplasia (BPD) in premature infants. Bronchoalveolar lavage fluid (BALF) samples were collected on the 3rd day of life from mechanically ventilated neonates with gestational age ≤ 30 weeks and analyzed without previous treatment (top-down proteomics) by reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. AAT fragments were identified by high-resolution LTQ Orbitrap XL experiments and the relative abundances determined by considering the extracted ion current (XIC) peak area. Forty preterm neonates were studied: 20 (50%) did not develop BPD (no-BPD group), 17 (42.5%) developed mild or moderate new-BPD (mild + moderate BPD group), and 3 (7.5%) developed severe new-BPD (severe BPD group). Eighteen fragments of AAT and a fragment of AAT oxidized at a methionine residue were identified: significantly higher values of AAT fragments 25-57, 375-418, 397-418, 144-171, and 397-418 with oxidized methionine were found in the severe BPD group. The significantly higher levels of several AAT fragments and of the fragment 397-418, oxidized in BALF of preterm infants developing BPD, underlie the central role of an imbalance between proteases and protease inhibitors in exacerbating lung injury and inducing most severe forms of BPD. The study has some limitations, and between them, the small sample size implies the need for further confirmation by larger studies.