12 research outputs found
Analysis of allergenic proteins by mass spectrometry
MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes.
Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergenâs structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data.
Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays.
Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins.
In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (ι-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked.
Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named âtargeted proteomicsâ was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample.
The procedure involves specific labelling and isolation of the specific peptide, named âproteotypicâ. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The âtargeted proteomicsâ procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research.
Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis.
The molecular properties of the complex formed by Îą-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation.
Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi dâelezione applicati per lâidentificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattivitĂ e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare lâinterazione con gli anticorpi specifici, invalidando i risultati.
Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per lâidentificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, lâidentificazione delle proteine mediante MS si basa sullâanalisi della sequenza amminoacidica di questâultime, mentre i saggi immunochimici sono strettamente dipendenti dallâintegritĂ della struttura tridimensionale della proteina antigenica.
Al fine di testare la validitĂ dellâapproccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte Îą-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualitĂ di questi anticorpi, in termini di specificitĂ di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattivitĂ . Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico.
Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata lâapplicazione di una tecnica detta âtargeted chromatographyâ. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso lâanalisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti âprototipiciâ. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dellâanello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando lâaumento di idrofobicitĂ e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinitĂ utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di âtargeted proteomicsâ è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per lâidentificazione di una proteina allergenica contenuta in un prodotto dolciario. Ă stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente lâanalisi di fingerprinting/MS che è solitamente utilizzata per lâidentificazione di proteine nei protocolli di proteomica.
Altre attivitĂ di ricerca. Durante il periodo di dottorato, ho avuto lâopportunitĂ di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato.
Sono state investigate le proprietĂ molecolari del complesso formato da Îą-lattalbumina con lâacido oleico. Il complesso appare interessante poichĂŠ ha mostrato avere tossicitĂ cellulare diretta selettivamente contro cellule tumorali. Ă stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che lâacido oleico interagisce anche con altre proteine, come lâapomioglobina.
La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola lâacido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dellâacido grasso e conseguentemente della sua proprietĂ citotossica. Ă in preparazione un articolo riguardante questi risultati.
Lâenterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attivitĂ antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticitĂ e una ridotta stabilitĂ alla denaturazione termica, ma mantiene la sua attivitĂ antibatterica., mentre i frammenti presentano unâattivitĂ ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per lâattivitĂ antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008)
Alpha-lactalbumin forms with oleic acid a high molecular weight complex displaying cytotoxic activity
\u3b1-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its \u3b1-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu
Quinolino[3,4-b]quinoxalines and pyridazino[4,3-c]quinoline derivatives: Synthesis, inhibition of topoisomerase II\u3b1, G-quadruplex binding and cytotoxic properties
The quinoline motif fused with other heterocyclic systems plays an important role in the field of anticancer drug development.
An extensive series of tetracyclic quinolino[3,4-b]quinoxalines N-5 or C-6 substituted with basic side chain and
a limited number of tricyclic pyridazino[4,3-c]quinolines N-6 substituted were designed, synthesized and evaluated for
topoisomerase II\u3b1 (Topo II\u3b1) inhibitory activity, ability to bind and stabilize G-quadruplex structures and cytotoxic properties
against two human cancer cell lines (HeLa and MCF-7). Almost all of the tested agents showed a high activity as
Topo II\u3b1 inhibitors and G-quadruplex stabilizers. Among all the derivatives studied, the quinolino[3,4-b]quinoxalines 11
and 23, N-5 and C-6 substituted respectively, stand out as the most promising compounds. Derivative 11 resulted a selective
binder to selected G-quadruplex sequences, while derivative 23 displayed the most interesting Topo II\u3b1 inhibitory
activity (IC50 = 5.14 \u3bcM); both showed high cytotoxic activity (IC50 HeLa = 2.04 \u3bcM and 2.32 \u3bcM, respectively)
Low Abundant <i>N</i>âlinked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites
Although wild-type
hen egg white lysozyme (HEL) is lacking the
consensus sequence motif NXÂ(S/T), in 1995 Trudel et al. (<i>Biochem.
Cell Biol.</i> <b>1995</b>, <i>73</i>, 307â309)
proposed the existence of a low abundant <i>N</i>-glycosylated
form of HEL; however, the identity of active glycosylation sites in
HEL remained a matter of speculation. For the first time since Trudelâs
initial work, we report here a comprehensive characterization by means
of mass spectrometry of <i>N</i>-glycosylation in wild-type
HEL. Our analytical approach comprised ZIC-HILIC enrichment of <i>N</i>-glycopeptides from HEL trypsin digest, deglycosylation
by <sup>18</sup>O/PNGase F as well as by various endoglycosidases,
and LCâMS/MS analysis of both intact and deglycosylated <i>N</i>-glycopeptides engaging multiple techniques of ionization
and fragmentation. A novel data interpretation workflow based on MS/MS
spectra classification and glycan database searching enabled the straightforward
identification of the asparagine-rich <i>N</i>-glycopeptide
[34â45] FESNFNTQATNR and allowed for compositional profiling
of its modifying <i>N</i>-glycans. The overall heterogeneity
profile of <i>N</i>-glycans in HEL comprised at least 26
different compositions. Results obtained from deglycosylation experiments
provided clear evidence of asparagine residues N44 and N39 representing
active glycosylation sites in HEL. Both of these sites do not fall
into any known <i>N</i>-glycosylation-specific sequence
motif but are localized in rarely observed nonconsensus sequons (NXN,
NXQ)
Characterization of linear forms of the circular enterocin AS-48 obtained by limited proteolysis
AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that circularization is not required for the bactericidal activity, but it is important to stabilize the native structure. Moreover, it is possible to reduce the sequence to a minimal AS-48 domain without causing inactivation of this bacteriocin
Hit Identification of a Novel Dual Binder for h-telo/c-myc G-Quadruplex by a Combination of Pharmacophore Structure-Based Virtual Screening and Docking Refinement
It is well known that G-quadruplexes are targets of great interest for their roles in crucial biological processes, such as aging and cancer. Hence, a promising strategy for anticancer drug therapy is the stabilization of these structures by small molecules. We report a high-throughput insilico screening of commercial libraries from several different vendors by means of a combined structure-based pharmacophore model approach followed by docking simulations. The compounds selected by the virtual screening procedure were then tested for their ability to interact with human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and fluorescence intercalator displacement. Our approach resulted in the identification of a 13-[(dimethylamino)methyl]-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one derivative as a novel promising stabilizer of G-quadruplex structures within the human telomeric and the c-myc promoter sequences
Exploring the Chemical Space of G-Quadruplex Binders: Discovery of a Novel Chemotype Targeting the Human Telomeric Sequence
Recent findings have unambiguously demonstrated that DNA G-rich sequences can adopt a G-quadruplex folding in living cells, thus further validating them as crucial targets for anticancer therapy. Herein, to identify new potent G4 binders as antitumor drug candidates, we have targeted a 24-nt G4-forming telomeric sequence employing a receptor-based virtual screening approach. Among the best candidates, in vitro binding experiments allowed identification of three novel G4 ligands. Among them, the best compound features an unprecedented binding selectivity for the human telomeric DNA G-quadruplex with no detectable binding for other G4-forming sequences present at different genomic sites. This behavior correlates with the detected ability to generate DNA damage response in tumor cells at the telomeric level and efficient antiproliferative effect on different tumor cell lines at low micromolar concentrations
Exploring the Chemical Space of GâQuadruplex Binders: Discovery of a Novel Chemotype Targeting the Human Telomeric Sequence
Recent
findings have unambiguously demonstrated that DNA G-rich
sequences can adopt a G-quadruplex folding in living cells, thus further
validating them as crucial targets for anticancer therapy. Herein,
to identify new potent G4 binders as antitumor drug candidates, we
have targeted a 24-nt G4-forming telomeric sequence employing a receptor-based
virtual screening approach. Among the best candidates, <i>in
vitro</i> binding experiments allowed identification of three
novel G4 ligands. Among them, the best compound features an unprecedented
binding selectivity for the human telomeric DNA G-quadruplex with
no detectable binding for other G4-forming sequences present at different
genomic sites. This behavior correlates with the detected ability
to generate DNA damage response in tumor cells at the telomeric level
and efficient antiproliferative effect on different tumor cell lines
at low micromolar concentrations