Publications by authors named "Marialaura Dilillo"

11 Publications

  • Page 1 of 1

Longitudinal Bottom-Up Proteomics of Serum, Serum Extracellular Vesicles, and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model.

Molecules 2021 Oct 2;26(19). Epub 2021 Oct 2.

Fondazione Pisana per la Scienza ONLUS, 56017 San Giuliano Terme, Italy.

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset.
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http://dx.doi.org/10.3390/molecules26195992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8512455PMC
October 2021

Synaptic Vesicles Dynamics in Neocortical Epilepsy.

Front Cell Neurosci 2020 10;14:606142. Epub 2020 Dec 10.

Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, United Kingdom.

Neuronal hyperexcitability often results from an unbalance between excitatory and inhibitory neurotransmission, but the synaptic alterations leading to enhanced seizure propensity are only partly understood. Taking advantage of a mouse model of neocortical epilepsy, we used a combination of photoconversion and electron microscopy to assess changes in synaptic vesicles pools . Our analyses reveal that epileptic networks show an early onset lengthening of active zones at inhibitory synapses, together with a delayed spatial reorganization of recycled vesicles at excitatory synapses. Proteomics of synaptic content indicate that specific proteins were increased in epileptic mice. Altogether, our data reveal a complex landscape of nanoscale changes affecting the epileptic synaptic release machinery. In particular, our findings show that an altered positioning of release-competent vesicles represent a novel signature of epileptic networks.
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http://dx.doi.org/10.3389/fncel.2020.606142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758433PMC
December 2020

Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model.

Sci Rep 2020 11 24;10(1):20498. Epub 2020 Nov 24.

Fondazione Pisana per la Scienza ONLUS, 56107, San Giuliano Terme, PI, Italy.

Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV's) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV's both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV's isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1).
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http://dx.doi.org/10.1038/s41598-020-77535-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686310PMC
November 2020

Quantitative Microproteomics Based Characterization of the Central and Peripheral Nervous System of a Mouse Model of Krabbe Disease.

Mol Cell Proteomics 2019 06 29;18(6):1227-1241. Epub 2019 Mar 29.

§Fondazione Pisana per la Scienza ONLUS, 56107 San Giuliano Terme, Pisa, Italy;

Krabbe disease is a rare, childhood lysosomal storage disorder caused by a deficiency of galactosylceramide beta-galactosidase (GALC). The major effect of GALC deficiency is the accumulation of psychosine in the nervous system and widespread degeneration of oligodendrocytes and Schwann cells, causing rapid demyelination. The molecular mechanisms of Krabbe disease are not yet fully elucidated and a definite cure is still missing. Here we report the first in-depth characterization of the proteome of the Twitcher mouse, a spontaneous mouse model of Krabbe disease, to investigate the proteome changes in the Central and Peripheral Nervous System. We applied a TMT-based workflow to compare the proteomes of the corpus callosum, motor cortex and sciatic nerves of littermate homozygous Twitcher and wild-type mice. More than 400 protein groups exhibited differences in expression and included proteins involved in pathways that can be linked to Krabbe disease, such as inflammatory and defense response, lysosomal proteins accumulation, demyelination, reduced nervous system development and cell adhesion. These findings provide new insights on the molecular mechanisms of Krabbe disease, representing a starting point for future functional experiments to study the molecular pathogenesis of Krabbe disease. Data are available via ProteomeXchange with identifier PXD010594.
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http://dx.doi.org/10.1074/mcp.RA118.001267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553931PMC
June 2019

Ultraviolet Photodissociation of ESI- and MALDI-Generated Protein Ions on a Q-Exactive Mass Spectrometer.

J Proteome Res 2019 01 4;18(1):557-564. Epub 2018 Dec 4.

Fondazione Pisana per la Scienza ONLUS , 56107 San Giuliano Terme, Pisa , Italy.

The identification of molecular ions produced by MALDI or ESI strongly relies on their fragmentation to structurally informative fragments. The widely diffused fragmentation techniques for ESI multiply charged ions are either incompatible (ECD and ETD) or show lower efficiency (CID, HCD), with the predominantly singly charged peptide and protein ions formed by MALDI. In-source decay has been successfully adopted to sequence MALDI-generated ions, but it further increases spectral complexity, and it is not compatible with mass-spectrometry imaging. Excellent UVPD performances, in terms of number of fragment ions and sequence coverage, has been demonstrated for electrospray ionization for multiple proteomics applications. UVPD showed a much lower charge-state dependence, and so protein ions produced by MALDI may exhibit equal propensity to fragment. Here we report UVPD implementation on an Orbitrap Q-Exactive Plus mass spectrometer equipped with an ESI/EP-MALDI. UVPD of MALDI-generated ions was benchmarked against MALDI-ISD, MALDI-HCD, and ESI-UVPD. MALDI-UVPD outperformed MALDI-HCD and ISD, efficiently sequencing small proteins ions. Moreover, the singly charged nature of MALDI-UVPD avoids the bioinformatics challenges associated with highly congested ESI-UVPD mass spectra. Our results demonstrate the ability of UVPD to further improve tandem mass spectrometry capabilities for MALDI-generated protein ions. Data are available via ProteomeXchange with identifier PXD011526.
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http://dx.doi.org/10.1021/acs.jproteome.8b00896DOI Listing
January 2019

Mass spectrometry imaging: How will it affect clinical research in the future?

Expert Rev Proteomics 2018 09 19;15(9):709-716. Epub 2018 Sep 19.

a Fondazione Pisana per la Scienza ONLUS , Pisa , Italy.

Introduction: Mass spectrometry imaging (MSI) is a label free, multiplex imaging technology able to simultaneously record the distributions of 100's to 1000's of species, and which may be configured to study metabolites, lipids, glycans, peptides, and proteins simply by changing the tissue preparation protocol. Areas covered: The capability of MSI to complement established histopathological practice through the identification of biomarkers for differential diagnosis, patient prognosis, and response to therapy; the capability of MSI to annotate tissues on the basis of each pixel's mass spectral signature; the development of reproducible MSI through multicenter studies. Expert commentary: We discuss how MSI can be combined with microsampling/microdissection technologies in order to investigate, with more depth of coverage, the molecular changes uncovered by MSI.
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http://dx.doi.org/10.1080/14789450.2018.1521278DOI Listing
September 2018

Characterization of Degraded Proteins in Paintings Using Bottom-Up Proteomic Approaches: New Strategies for Protein Digestion and Analysis of Data.

Anal Chem 2018 06 21;90(11):6403-6408. Epub 2018 May 21.

Department of Chemistry and Industrial Chemistry , University of Pisa , Pisa , Italy 56124.

Chemical hydrolysis assisted by microwave irradiation has been proposed as an alternative method for the analysis of proteins in highly insoluble matrices. In this work, chemical hydrolysis was applied for the first time to detect degraded proteins from paintings and polychromies. To evaluate the performance of this approach, the number of identified peptides, protein sequence coverage (%), and PSMs were compared with those obtained using two trypsin-based proteomics procedures used for the analysis of samples from cultural heritage objects. It was found that chemical hydrolysis allowed the successful identification of all proteinaceous materials in all paint samples analyzed except for egg proteins in one extremely degraded sample. Moreover, in one sample, casein was only identified by chemical digestion. In general, chemical hydrolysis identified more peptides, more PSM's, and greater sequence coverage in the samples containing caseins, and often also in animal glue, highlighting the great potential of this approach for the rapid digestion and identification of insoluble and degraded proteins from the field of the cultural heritage.
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http://dx.doi.org/10.1021/acs.analchem.8b00281DOI Listing
June 2018

Mass Spectrometry Imaging, Laser Capture Microdissection, and LC-MS/MS of the Same Tissue Section.

J Proteome Res 2017 08 5;16(8):2993-3001. Epub 2017 Jul 5.

Fondazione Pisana per la Scienza ONLUS , 56121 Pisa, Italy.

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.
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http://dx.doi.org/10.1021/acs.jproteome.7b00284DOI Listing
August 2017

Design and Performance of a Novel Interface for Combined Matrix-Assisted Laser Desorption Ionization at Elevated Pressure and Electrospray Ionization with Orbitrap Mass Spectrometry.

Anal Chem 2017 07 28;89(14):7493-7501. Epub 2017 Jun 28.

Fondazione Pisana per la Scienza ONLUS , 56121 Pisa, Italy.

Matrix-Assisted Laser Desorption Ionization, MALDI, has been increasingly used in a variety of biomedical applications, including tissue imaging of clinical tissue samples, and in drug discovery and development. These studies strongly depend on the performance of the analytical instrumentation and would drastically benefit from improved sensitivity, reproducibility, and mass/spatial resolution. In this work, we report on a novel combined MALDI/ESI interface, which was coupled to different Orbitrap mass spectrometers (Elite and Q Exactive Plus) and extensively characterized with peptide and protein standards, and in tissue imaging experiments. In our approach, MALDI is performed in the elevated pressure regime (5-8 Torr) at a spatial resolution of 15-30 μm, while ESI-generated ions are injected orthogonally to the interface axis. We have found that introduction of the MALDI-generated ions into an electrodynamic dual-funnel interface results in increased sensitivity characterized by a limit of detection of ∼400 zmol, while providing a mass measurement accuracy of 1 ppm and a mass resolving power of 120 000 in analysis of protein digests. In tissue imaging experiments, the MALDI/ESI interface has been employed in experiments with rat brain sections and was shown to be capable of visualizing and spatially characterizing very low abundance analytes separated only by 20 mDa. Comparison of imaging data has revealed excellent agreement between the MALDI and histological images.
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http://dx.doi.org/10.1021/acs.analchem.7b01168DOI Listing
July 2017

In-Source Decay and Pseudo-MS of Peptide and Protein Ions Using Liquid AP-MALDI.

J Am Soc Mass Spectrom 2016 12 17;27(12):2075-2079. Epub 2016 Oct 17.

Fondazione Pisana per la Scienza ONLUS, Pisa, Italy.

Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS method. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1511-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088222PMC
December 2016

Discovering "The Italian Flag" by Fernando Melani (1907-1985).

Spectrochim Acta A Mol Biomol Spectrosc 2016 Nov 25;168:52-59. Epub 2016 May 25.

Istituto di Chimica dei Composti Organometallici - Consiglio Nazionale delle Ricerche (ICCOM-CNR), via Giuseppe Moruzzi 1, 56124 Pisa, Italy.

In the occasion of the celebrations for the 150th anniversary of the founding of Italy (1861-2011), it was decided to analyse the artwork "The Italian Flag" (La Bandiera Italiana) created by the artist Fernando Melani (Pistoia, 1907-1985), one of the precursors of the Poor Art artistic movement in Italy. This project is a follow-up to a previous study which was mainly focused on the pigments and dyes found in his home-studio. The main goal of this paper is to identify a correct diagnostic plan, based on the use of a combination of non-invasive and micro-invasive methodologies, in order to determine the state of preservation and define the best conservation procedures for a contemporary artwork. Visible, infrared and infrared false colour images as well as the Fibre Optic Reflectance Spectroscopy (FORS) technique were applied in situ to analyse The Italian Flag. Laser Induced Breakdown Spectroscopy (LIBS), Fourier Transform Infrared (FT-IR) and micro-Raman spectroscopies, Pyrolysis-Gas Chromatography/Mass Spectroscopy (Py-GC/MS), High Performance Liquid Chromatography with Diode Arrays Detection (HPLC-DAD) and Mass Spectrometric Detection (HPLC-ESI-Q-ToF) were all applied to three small samples detached from the three painted (green-blue, white and red-yellow, respectively) areas of the flag. The combination of the data obtained with all these techniques made possible a comprehensive understanding of both the chemical composition and physical behaviour of the materials used by the artist and supported curators in defining the preventive conservation of this artwork.
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http://dx.doi.org/10.1016/j.saa.2016.05.027DOI Listing
November 2016
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