Targeted tandem mass spectrometry strategies to quantify proteins biomarkers of inflammatory diseases

Abstract Mass spectrometry is a sensitive technique used to detect, identify and quantitate molecules based on their mass-to-charge (m/z) ratio in simple and complex mixtures. Originally developed almost 100 years ago to measure elemental atomic weights and the natural abundance of specific isotopes, MS was first used in the biological sciences to trace heavy isotopes through biological systems. The following PhD project was involved in the application of advanced methods of mass spectrometry in Multiple Reaction Monitoring ion mode (MRM) into different metabolomics and proteomics research areas. For the most part, the studies conducted over the past three years have had a single common thread: development and application of MRM mass spectrometry methods for the identification and quantification of targeted metabolites and/or proteins involved in inflammatory processes. It is clear that, for each project, we started from the study of the different biological matrices to find the most effective extraction strategy for the target analytes and subsequent steps have covered in-depth literature studies to identify the best condition to perform chromatographic separation and the subsequent optimization of instrumental parameters. The three main areas of application explored in these years concerned the following three points: 1. MRM/MS analysis of metabolites In collaboration with Prof. Greco, Prof. Auricchio and Prof. Ruoppolo of the Department of Translational Medical Sciences and Department of Molecular Medicine and Medical Biotechnology of the University of Naples Federico II. The project provided for the development of a method that allow the investigation of the lipidomic profile of genetically predisposed children to celiac disease in order to identify potential molecular biomarkers for disease prediction, first of all, in genetically predisposed patients but also for subjects whose clinical history is unknown. Serum samples of two cohorts: 23 children who became coeliac and 23 not yet (used as control), do share a similar genetic background, since they come from families with one celiac proband and bear the specific HLA haplotype (DQ2 or DQ8). It does appear that the genetic profile may not explain fully the great differences found between the two cohorts. The developed MRM method allowed to monitor and quantify 83 different classes of analytes and allowed us to identify some classes of lipids as putative molecular biomarkers by comparing the results obtained from the analysis of the samples of serum collected at 4 months, before introduction of gluten, at 12 months, with the introduction of gluten in the diet of the child and a t> 12 months for children who have been diagnosed with celiac disease. 2. MRM/MS analysis of proteins. A peculiar feature of a MRM method is the ability to monitor multiple precursor ion-product ion transitions. This greatly increase the selectivity and specificity of the analysis and this represents a huge advantage in the proteomic field because each target analyte, in this case peptide, it can be identified within complex mixtures (such as biological fluids) by monitoring transitions closely related to its own amino acid sequence. Different biological aspects were investigated: - The project, in collaboration with Prof. Piccoli and Dr. Arciello of the Department of Chemical Sciences of the University of Naples Federico II, involved the development and optimization of an MRM method for the quantification of proteins involved in inflammatory processes: TNF -α, INF-γ, IL-8 and IL-10 in THP-I cell samples. In particular, differentiated cells have been treated with LPS, a well-known endotoxin, to stimulate the onset of an inflammatory process. A time course analysis was performed on differentiated and stimulated cells with LPS for 2h, 4h, 6h, 9h and 24h. These analyses allowed to monitor the variation in protein expression during the whole inflammatory process, in both acute and late phase of the inflammation and the obtained data are consistent with published works. Quantitative analysis was conducted using the external standard method. In order to increase the selectivity and the specificity of the method, for each target protein, two or three peptides have been identified thanks to the aid of bioinformatic software which have a unique amino acid sequence and can be used as a stoichiometric representation of the protein in the quantitative analysis. - Project in collaboration with prof. Francisco Blanco and Dr. Cristina Ruiz-Romero of the INIBIC Biomedical Research Institute of A Coruña, Spain. At this research institute I spent six months for the foreign period of the PhD. In these months I have worked on both the optimization of a SIS-MRM/MS method for the quantification of proteins involved in rheumatoid arthritis (RA) pathogenesis and the application of this method to a cohort of 80 serum samples of subjects whom RA has been diagnosed. Quantitative analysis was performed using the internal standard method: stable isotope labelled standard peptides (SIS) were used. The target peptides belong to proteins that were statistically significant (p <0.05) in previous experiments of 8-plex iTRAQ and large-scale proteomics. RA diagnosis is complex and nowadays it is carried out by putting together radiographic data, DAS 28 and serum parameters such as C-reactive protein, rheumatoid factor (RF) and anti-citrullinated antibodies (ACPA) levels. The main problem in diagnostics is incurring false positives, as in the case of RF that shows the same trend also in other diseases like: chronic hepatitis, chronic viral infections, leukaemia, dermatomyositis, mononucleosis, scleroderma, Hashimoto's thyroiditis, systemic lupus erythematosus and Sjögren's syndrome. First step involved the development of the method, the validation of the selected transitions and the choice of the concentration of labelled peptides to be added to the real samples to obtain the best signal/noise ratio. Reverse calibration curves for each heavy target peptide were realized and analytical parameters Detection limit (LOD), Quantification limit (LOQ) and linearity range were calculated. The developed method was used for the identification and quantification of the 10 target proteins in a cohort of 80 samples of sera from RA patients subdivided into 4 sub-groups based on rheumatoid factor (RF) and anti-citrullinated antibodies (ACPA) values. The four sub-groups were: RF-/ACPA-, RF-/ACPA+, RF+/ACPA- and RF+/ACPA+. Data analysis allowed to select some of the proteins monitored for the subsequent steps of method validation by using complementary techniques such as ELISA immunoassay. 3. MRM/MS analysis for discovery. - An interesting application of mass spectrometry in MRM ion mode was in forensic field for the identification of biomarker proteins of biological fluids. This project was carried out in collaboration with Ten.Col. Peluso of the RIS department of the Carabinieri in Rome. The basic idea was to exploit the potential of mass spectrometry in MRM ion mode to identify the nature of biological traces found at a crime scene from which the DNA was extracted. In this way it is possible to conduct complementary investigations on both the identity of the suspect and the sequence of events that lead to the crime. Presumptive and confirmatory tests are needed to be absolutely sure of the identity of the biological fluid found at a crime scene. These tests suffer from limitations due, above all, to poor specificity and to the necessity to conduct cascade tests to evaluate the nature of the trace. The developed MRM method allows to overcome these limitations as it allows to discriminate between four biological matrices: blood, saliva, seminal fluid and urine through a single analysis and through a single sample treatment, which involves hydrolysis with trypsin, to carry out the extraction DNA and the subsequent proteomic analysis. - Project in collaboration with Prof Nicosia from Department of Molecular Medicine and Medical Biotechnologies of the University of Naples Federico II for the identification of proteins in HCT116 cells (human colorectal tumour cells) deriving from alternative splicing processes and which could be closely related to the onset and progression of this type of cancer. Previous studies performed on mRNA have shown that such alternative splicing phenomena lead to proteins that have a mutated sequence to C-ter. This sequence was used for setting up of the MRM / MS method thanks to the high specificity and selectivity that derives from the use of mass spectrometry in this mode, but above all the high sensitivity to which it is possible to arrive (amol/μL). A method was developed to identify the presence of 5 mutated proteins using the mutated C-ter aminoacidic sequence as molecular target and it was possible to verify the presence of these mutated sequences confirming the translation of these proteins. Finally, for the method validation HT 29 and LS147T cell lines were used. The results seem promising, in all the selected cell lines the mutated proteins were detected. Further developments concern the implementation of the MRM / MS method developed with the use of isotopically labelled peptides to validate and perform quantitative analysis. Results obtained in the present PhD thesis show the broad applicability of the MRM/MS methodology. This strategy was effective both during the discovery phase and for the quantitative analysis of metabolites and proteins, demonstrating high sensitivity, selectivity and specificity. The chance to simultaneously analyse a panel of numerous analytes allows to optimize analysis times and costs. Finally, the "multiple" ability of this method allows all methods to be implemented by inserting the characteristic transitions of heavy isotopically labelled standards that have the same chemical and physical properties of the target molecules but different m/z ratio. The use of an internal standard is fundamental to evaluate the matrix effect, the efficiency of an extraction methodology and the identification of an analyte in complex samples. Subsequent steps for the presented projects are mainly focused on the implementation of the developed MRM methods with isotopically labelled standards, the validation of the obtained results by complementary techniques (ELISA) and the development of kits that can be used in clinical practice for diagnostics or follow-up of patients suffering from various diseases or in forensic investigations

Inflammation protein quantification by multiple reaction monitoring mass spectrometry in lipopolysaccharide-stimulated THP-1 cells

Rationale: Inflammation is a cascade of events mediated by a cytokine network triggering the cellular response. In order to monitor the modulation of the crucial inflammatory proteins, e.g., Tumour Necrosis Factor-α (TNF-α), Interferon-γ (INF-γ), Interleukin-8 (IL-8) and Interleukin-10 (IL-10), upon stimulation with endotoxins, differentiated and undifferentiated THP-1 cells were treated with lipopolysaccharides (LPSs) from E. coli, key cell wall components of Gram-negative bacteria. Methods: The multiple reaction monitoring mass spectrometry (MRM-MS) method was optimized by using the standard proteins to be quantified, in order to construct external calibration curves and define the analytical parameters. The developed method was used to quantify the above-mentioned inflammatory proteins in THP-1 differentiated cells upon stimulation with LPSs with high accuracy, sensitivity, and robustness. Results: The analysis of such proteins in MRM mode allowed the kinetics of stimulation along the time up to 24 h to be followed and the MS results were found to be comparable with those obtained by Western-blotting. A significant increase in TNF-α release triggered a cascade mechanism leading to the production of INF-γ and IL-8. IL-10, instead, was found to be constant throughout the process. Conclusions: The developed MRM-MS method allowed the quantification of TNF-α, INF-γ, IL-8 and IL-10 along a time-course from 2 to 24 h. Hence, a trace of the kinetics of the inflammatory response in THP-1 cells upon stimulation with E. coli LPSs was obtained. Finally, the extensibility of the developed MRM method to serum samples and other matrices demonstrated the versatility of the approach and the possibility to quantify multiple target proteins in different biological samples by using a few microliters in a single analysis

The Role of NGF and Its Receptor TrKA in Patients With Erectile Dysfunction

The aim of our study was to investigate the plasma NGF concentration and TrkA/p75NTR receptor expression on white blood cells (WBCs), in peripheral and corpus cavernosum blood isolated from patients with erectile dysfunction and metabolic syndrome (ED/MetS). This was a pilot case–control study. Inclusion criteria were as follows: men 18–65 years with ED and MetS and healthy subjects. The first sampling was performed at the level of the cubital vein (VC). Subsequently, 20 μg of intracavernous alprostadil was administered, and a second blood draw from the corpora cavernosa (CC) was performed once erection was achieved. Subsequently, the third blood sample was repeated at the level of the VC. We enrolled 8 cases with ED/MetS and 8 controls. There was no significant difference between the case and control group in terms of mean age (49.3 ± 5.9 and 53.13 ± 8.9, respectively). The case group had a lower IIEF score compared to the control group (14 ± 3.2 versus 27.3 ± 2.1; p &lt; 0.05). Decreased NGF and TrKA expression on WBC and thiols were found in the plasma of ED/MetS patients compared to control. The study showed that patients with ED/MetS had a decrease in plasma NGF and thiol concentration, and they had a decrease in TrKA expression on WBCs

Identification of proteinaceous binders in paintings: A targeted proteomic approach for cultural heritage

Abstract Identification of proteins in paintings and polychrome objects is a challenge, which requires the development of tailored analytical approaches. In the present study, a targeted proteomics approach was developed for discriminating among the three most common proteinaceous materials used as paint binders, i.e. milk, egg, and animal glue. In this study a specific database of peptides was created based on tandem MS analyses of tryptic digests of several paint samples collected from a variety of art objects of different ages and conservation conditions. Specific peptide markers of each protein were then selected and monitored by LC-MSMS in Multiple Reaction Monitoring (MRM) ion mode, together with their specific precursor ion-product ion transitions, as defined by their unique amino acid sequence. The developed method enabled a sensitive and reliable detection of the target peptides in a selection of case studies, leading to the unambiguous identification of the proteins used as paint binders. The method showed greatly increased sensitivity compared to currently available strategies

Targeted Phospholipidomic Analysis of Synovial Fluid as a Tool for Osteoarthritis Deep Phenotyping

[Abstract] Objective. The aim of this study was to carry out a targeted phospholipidomic analysis on synovial fluid (SF) from patients with different grades of osteoarthritis (OA) and controls, in order to search for specific phospholipid profiles that may be useful for the deep phenotyping of this disease. Design. Multiple reaction monitoring-mass spectrometry (MRM/MS) was applied to explore the potential phospholipidomic differences in the SF of knee OA patients (n ​= ​15) (subclassified into early- and late-stage OA) and non-OA controls (n ​= ​4). Multivariate statistical analyses conducted by partial least squares discriminant analysis (PLS-DA) and hierarchical clustering analysis (HCA) were performed to identify significantly altered phospholipids in OA, characterize phospholipidomic profiles associated with the radiographic stage of the disease and describe potential endotypes at early stages. Results. Significant discrimination of phospholipid profiles between non-OA controls and the early- and late-stage OA groups were found by PLS-DA and HCA. Compared to SF from non-OA controls, OA patients showed higher levels of most quantified phospholipid species, including phosphatidylcholines (PC), phosphatidylserines and phosphatidylinositols. Furthermore, several PC species showed significant differences in abundance between the two OA subgroups and were negatively correlated with cartilage damage. Finally, two distinct endotypes of early-stage OA were identified based on the phospholipidomic profile of SF. Conclusions. Our data provides a novel insight into the phospholipid profiles of OA synovial fluid, revealing specific alterations associated with the radiographic stage of the disease. This targeted phospholipidomic profiling also facilitated the characterization of two different OA endotypes at early stages of the disease.This work is supported by grants from Fondo Investigación Sanitaria-Spain (PI16/02124, PI17/00404, PI19/01206, PI20/00793 and RETIC-RIER-RD16/0012/0002), integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research - European Regional Development Fund (FEDER) “A way of making Europe”. This study is also supported by AE CICA-INIBIC (ED431E 2018/03) and grants IN607A 2017/11, IN607A 2021/7 and IN607D 2020/10 from Axencia Galega de Innovacion - Xunta de Galicia. The Biomedical Research Networking Center (CIBER) is an initiative from Instituto de Salud Carlos III (ISCIII). The Proteomics Unit of GIR belongs to ProteoRed, PRB3- ISCIII (PT17/0019/0014)Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN607A 2017/11Xunta de Galicia; IN607A 2021/7Xunta de Galicia; IN607D 2020/1

Malvidin and cyanidin derivatives from açai fruit (Euterpe oleracea Mart.) counteract UV-A-induced oxidative stress in immortalized fibroblasts

UV-A radiations are known to induce cellular oxidative stress, leading to premature skin aging. Consumption of açai fruit (Euterpe oleracea Martius) is known to have many health benefits due to its high level of antioxidants. Herein, we analyzed the ability of phenolic compounds extracted from this fruit to attenuate UV-A-induced oxidative stress in immortalized fibroblast. A methanol/water açai extract was fractionated by HPLC and each fraction tested for anti-oxidant stress activity. Immortalized fibroblasts were pre-incubated with açai fractions and then exposed to UV-A radiations. Açai extract was found to be able to strongly protect cells from oxidative stress. In particular, reactive oxygen species (ROS) production, GSH depletion, lipid peroxidation and no increase in the phosphorylation levels of proteins involved in the oxidative stress pathway was observed in cells pre-incubated with the extract and then irradiated by UV-A. Mass spectrometry analyses of HPLC fractionated extract led us to the identification of malvidin and cyanidin derivatives as the most active molecules able to counteract the negative effects induced by UV-A irradiation. Our results indicate, for the first time, that açai fruit is a valuable natural source for malvidin and cyanidin to be used as anti-stress molecules and represent good candidates for dietary intervention in the prevention of age related skin damage

The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins

: Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans

COVID-19 disease in clinical setting: impact on gonadal function, transmission risk, and sperm quality in young males.

Abstract Objectives We want to evaluate the possible presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in semen samples and semen quality, looking for a possible relationship between the infectious disease and fertility. Methods In this prospective study, we enrolled 15 consecutive men (age 18–50 years) with positive oropharyngeal swab to SARS-CoV-2 and classified, according to WHO criteria, in mild to moderate disease. A semen sample was collected to detect SARS-CoV viral RNA by the automated Real-Time PCR ELITe InGenius® system and the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup, France). Analysis of semen characteristics was performed according to WHO laboratory manual 5th ed. for the examination and processing of human semen. Blood samples for the dosage of hormonal assay, procalcitonin, interleukin 6, C-reactive protein were obtained. Results SARS-CoV-2 RNA has not been detected in semen samples from any of the subjects analysed. Sperm analysis exhibited abnormal seminal values in 14 out of 15 patients (93.3%). Furthermore, no difference was detected regarding sperm quality between mild and moderate SARS-CoV-2 patients. No alteration in the inflammatory indices was observed in the studied population, as well gonadotropins and testosterone levels. Conclusions COVID patients studied exhibits alteration of the seminal fluid both in microscopic and macroscopic characteristics such as hypoposia and increased viscosity, which have not been detected in previous studies. The presence of viral RNA within the seminal fluid was excluded

Association of the serological status of rheumatoid arthritis patients with two circulating protein biomarkers: a useful tool for precision medicine strategies

[Abstract] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and presence of systemic autoantibodies, with a great clinical and molecular heterogeneity. Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are routinely used for the diagnosis of RA. However, additional serological markers are needed to improve the clinical management of this disease, allowing for better patient stratification and the desirable application of precision medicine strategies. In the present study, we investigated those systemic molecular changes that are associated with the RF and ACPA status of RA patients. To achieve this objective, we followed a proteomic biomarker pipeline from the discovery phase to validation. First, we performed an iTRAQ-based quantitative proteomic experiment on serum samples from the RA cohort of the Hospital of Santiago de Compostela (CHUS). In this discovery phase, serum samples from the CHUS cohort were pooled according to their RF/ACPA status. Shotgun analysis revealed that, in comparison with the double negative group (RF-/ACPA-), the abundance of 12 proteins was altered in the RF+/ACPA+ pool, 16 in the RF+/ACPA- pool and 10 in the RF-/ACPA+ pool. Vitamin D binding protein and haptoglobin were the unique proteins increased in all the comparisons. For the verification phase, 80 samples from the same cohort were analyzed individually. To this end, we developed a Multiple Reaction Monitoring (MRM) method that was employed in a comprehensive targeted analysis with the aim of verifying the results obtained in the discovery phase. Thirty-one peptides belonging to 12 proteins associated with RF and/or ACPA status were quantified by MRM. In a final validation phase, the serum levels of alpha-1-acid glycoprotein 1 (A1AG1), haptoglobin (HPT) and retinol-binding protein 4 (RET4) were measured by immunoassays in the RA cohort of the Hospital of A Coruña (HUAC). The increase of two of these putative biomarkers in the double seropositive group was validated in 260 patients from this cohort (p = 0.009 A1AG1; p = 0.003 HPT). The increased level of A1AG1 showed association with RF rather than ACPA (p = 0.023), whereas HPT showed association with ACPA rather than RF (p = 0.013). Altogether, this study has allowed a further classification of the RA seropositive patients into two novel clusters: RF+A1AG+ and ACPA+HPT+. The determination of A1AG1 and HPT in serum would provide novel information useful for RA patient stratification, which could facilitate the effective implementation of personalized medicine in routine clinical practice.Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; PI19/01206Instituto de Salud Carlos III; PI20/00793Instituto de Salud Carlos III; RD16/0012/0002Instituto de Salud Carlos III; RD21/0002/0009Xunta de Galicia; IN607A2021/07Xunta de Galicia; IN607D2020/1

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, \u3b3-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients