Pushing the limits of two-dimensional mass spectrometry

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) is a data independent tandem mass spectrometry technique that allows direct correlation between precursor and fragment ions without the necessity of any sort of isolation prior to fragmentation. Two-dimensional mass spectrometry (2D MS) experiments were conceived in the 1980s from inspiration of two-dimensional nuclear magnetic resonance (2D-NMR), but the development of the technique was stopped because of the insufficient computational capabilities of that time. In 2010, with the progress in computer technology and the improvements in FT-ICR MS instrumentation and methods, the interest in developing 2D MS was renewed. Since then, 2D FT-ICR MS showed to be a valuable platform for the analysis of small molecules, macromolecules such as peptides and glycopeptides, and complex mixtures deriving from tryptic digestion of proteins of increasing sizes. All the proteomics studies performed with 2D MS used the bottom-up (BUP) approach. The main goal of this Ph.D. research work was to develop two-dimensional mass spectrometry for the challenges of contemporary mass spectrometry, therefore pushing the limits of the technique towards the most interesting, cutting-edge research grounds, by optimising the technique, developing 2D MS analysis methods, and finally constructing improved instrumentation act to better perform 2D MS experiments. On this purpose, the rising importance of top-down proteomics (TDP) led to the development of 2D FT-ICR MS for the analysis of intact proteins. To realise this first project, 2D MS was optimised for TDP analysis using calmodulin (CaM) as a model, and infrared multiphotondissociation (IRMPD) as fragmentation technique. The study compared the two-dimensional BUP and TDP analysis of CaM with standard one-dimensional tandem mass spectrometry, and showed that the use of 2D MS allowed to obtain comparable cleavage coverages, with a consistent saving in sample and time. The study affirmed the suitability of 2D MS for top-down proteomics, still leaving space for more developments allowing its optimisation. The total cleavage coverage of CaM was later implemented with the use of BUP 2D electron-capture dissociation (ECD) MS. In order to improve the cleavage coverages obtained in top-down two-dimensional mass spectrometry, another technique was developed, called MS/2D MS. The technique makes use of an extra fragmentation step before two-dimensional mass spectrometry, achieved through external isolation of a charge state of interest, and collisional fragmentation in a hexapole-based collision cell before the ions are sent to the ICR-cell. The output of MS/2D MS is a single 2D mass spectrum with information equivalent to MS3 about the entire mixture. The technique has been successfully developed using ubiquitin (Ubi) and calmodulin as models, and showed new uses of the features exclusive of 2D MS, such as the use of precursor ion scans to assign the protein terminus of many precursors ions at once, and the analysis of internal fragments formed by the first dissociation. Furthermore, as long as extra fragmentation steps can be performed, a deeper investigation of the analytes can be achieved, in an experiment collectively called MSn /2D MS. After the development of 2D MS for TDP, another method was developed, implementing the use of two-dimensional mass spectrometry for another type of complex mixtures: polymers. An extensive 2D MS study was performed on a simple mixture of homopolymers such as PMMA, and on two complex mixtures commonly used as drug excipients, polysorbate 80 and TPGS. The study demonstrated again that single 2D MS experiments give results equivalent to multiple MS/MS experiments targeted at the different precursors. Furthermore, it allowed the high resolution tandem mass spectrometry of polysorbate 80, representing the first kind of study of that type because of the challenges of isolating single analytes from the polymer mixture without the use of a previous separation, such as chromatography. The use of neutral-loss lines was particularly useful in this study, allowing to discriminate between oleated, linoleated, and non-esterified species. Finally, 2D mass spectra of polymers showed characteristic diagonal lines, specific for each polymeric mixture, representing some sort of fingerprint of the mixture. The last part of the thesis is dedicated to the optimisation of 2D FT-ICR MS for ECD MS, the application of the technique to another class of compounds, glycans and glycan-derivatives, and finally to the development of a more capacious ICR-cell. The research performed in this thesis showed that 2D FT-ICR MS is a viable data independent mass spectrometry technique for top-down proteomics and polymer analysis; that the technique is fast, and it possesses unique features that improve ion assignments, such as precursor ion scans and neutral-loss lines. Finally, it opens for two-dimensional mass spectrometry the path to glycomics and to an improved 2D MS analysis of even more complex mixtures, thanks to the development of a novel ICR-cell

Application of tandem two-dimensional mass spectrometry for top-down deep sequencing of calmodulin

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%

AIF-EL - An OWL2-EL-compliant AIF ontology

This paper briefly describes AIF-EL, an OWL2-EL compliant ontology for the Argument Interchange Format.</p

Design, Synthesis, and in Vitro Evaluation of 4-(4-Hydroxyphenyl)piperazine-Based Compounds Targeting Tyrosinase

Melanin biosynthesis is enzymatically regulated by tyrosinase (TYR, EC 1.14.18.1), which is efficiently inhibited by natural and synthetic phenols, demonstrating potential therapeutic application for the treatment of several human diseases. Herein we report the inhibitory effects of a series of (4-(4-hydroxyphenyl)piperazin-1-yl)arylmethanone derivatives, that were designed, synthesised and assayed against TYR from Agaricus bisporus (AbTYR). The best inhibitory activity was predominantly found for compounds bearing selected hydrophobic ortho-substituents on the aroyl moiety (IC50 values in the range of 1.5–4.6 μM). They proved to be more potent than the reference compound kojic acid (IC50=17.8 μM) and displayed competitive mechanism of inhibition of diphenolase activity of AbTYR. Docking simulation predicted their binding mode into the catalytic cavities of AbTYR and the modelled human TYR. In addition, these compounds displayed antioxidant activity combined with no cytotoxicity in MTT tests. Notably, the best inhibitor affected tyrosinase activity in α-MSH-stimulated B16F10 cells, thus demonstrating anti-melanogenic activity

2D FT-ICR MS of Calmodulin : a top-down and bottom-up approach

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments

CT Perfusion as a Predictor of the Final Infarct Volume in Patients with Tandem Occlusion

Background: CT perfusion (CTP) is used in patients with anterior circulation acute ischemic stroke (AIS) for predicting the final infarct volume (FIV). Tandem occlusion (TO), involving both intracranial large vessels and the ipsilateral cervical internal carotid artery could generate hemodynamic changes altering perfusion parameters. Our aim is to evaluate the accuracy of CTP in the prediction of the FIV in TOs. Methods: consecutive patients with AIS due to middle cerebral artery occlusion, referred to a tertiary stroke center between March 2019 and January 2021, with an automated CTP and successful recanalization (mTICI = 2b - 3) after endovascular treatment were retrospectively included in the tandem group (TG) or in the control group (CG). Patients with parenchymal hematoma type 2, according to ECASS II classification of hemorrhagic transformations, were excluded in a secondary analysis. Demographic, clinical, radiological, time intervals, safety, and outcome measures were collected. Results: among 319 patients analyzed, a comparison between the TG (N = 22) and CG (n = 37) revealed similar cerebral blood flow (CBF) &gt; 30% (29.50 +/- 32.33 vs. 15.76 +/- 20.93 p = 0.18) and FIV (54.67 +/- 65.73 vs. 55.14 +/- 64.64 p = 0.875). Predicted ischemic core (PIC) and FIV correlated in both TG (tau = 0.761, p &lt; 0.001) and CG (tau = 0.315, p = 0.029). The Bland-Altmann plot showed agreement between PIC and FIV for both groups, mainly in the secondary analysis. Conclusion: automated CTP could represent a good predictor of FIV in patients with AIS due to TO

Renal Function Outcomes in Metastatic Non-Small-Cell Lung Carcinoma Patients Treated with Chemotherapy or Immune Checkpoint Inhibitors: An Unexpected Scenario

Immune checkpoint inhibitors (ICIs) and platinum-based chemotherapy (CT) are effective therapeutic agents for the palliative treatment of metastatic non-small-cell lung cancer (NSCLC); the aim of our study was to investigate the acute and chronic renal toxicities in this setting. We collected data on 292 patients who received cisplatin (35%), carboplatin-based regimens (25%), or ICI monotherapy (40%). The primary and secondary outcomes were compared to the acute kidney injury (AKI) rate and the mean estimated GFR (eGFR) decay between groups, respectively, over a mean follow-up duration of 15 weeks. We observed 26 AKI events (8.9%), mostly stage I AKI (80.7%); 15% were stage II AKI, 3.8% were stage III, and none required renal replacement therapy or ICU admission. The AKI rates were 10.9%, 6.8%, and 8.9% for the cisplatin, carboplatin, and ICI groups, respectively, and no significant differences were observed between the groups (p = 0.3). A global mean eGFR decay of 2.2 mL/min was observed, while for the cisplatin, carboplatin, and ICI groups, the eGFR decay values were 2.3 mL/min, 1.1 mL/min, and 3.5 mL/min, respectively. No significant differences were observed between the groups. Cisplatin/carboplatin-based CT and ICIs resulted in a similar incidence of AKI and eGFR decay, suggesting the safety of their cautious use, even in CKD patients

Strangeness, Charm and Beauty in Quark Matter: SQM 2007 Experimental Overview

This paper aims at providing an experimental overview of the Strangeness in Quark Matter 2007 ConferenceComment: Proceedings of Strangeness in Quark Matter 2007, submitted to Journal of Physics

Diabetes mellitus induces bone marrow microangiopathy

Objective-The impact of diabetes on the bone marrow (BM) microenvironment was not adequately explored. We investigated whether diabetes induces microvascular remodeling with negative consequence for BM homeostasis. Methods and Results-We found profound structural alterations in BM from mice with type 1 diabetes with depletion of the hematopoietic component and fatty degeneration. Blood flow (fluorescent microspheres) and microvascular density (immunohistochemistry) were remarkably reduced. Flow cytometry verified the depletion of MECA-32(+) endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker beta-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage(-) c-Kit(+) Sca-1(+) cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage(-) c-Kit(+) Sca-1(+) cells predominantly in the low-perfused part of the marrow. Cell depletion was associated to increased oxidative stress, DNA damage, and activation of apoptosis. Boosting the antioxidative pentose phosphate pathway by benfotiamine supplementation prevented microangiopathy, hypoperfusion, and lineage(-) c-Kit(+) Sca-1(+) cell depletion. Conclusion-We provide novel evidence for the presence of microangiopathy impinging on the integrity of diabetic BM. These discoveries offer the framework for mechanistic solutions of BM dysfunction in diabetes. (Arterioscler Thromb Vasc Biol. 2010;30:498-508.