My thesis focused on two aspects of proteomics applications in the vaccinology field that requires supportive bioinformatic applications. The first one refers to data management. Computational methods were applied for the identification of an innovative protocol making use of trypsin in sub-minute amount for short digestion times to access to the Pseudomonas aeruginosa periplasmic proteins, without causing the lysis of the whole bacteria, which could have provoked the leakage of other bacterial cellular fractions. The tools CELLO and PSORTb were first used to predict the localization of the identified proteins and therefore to discriminate the presence of contaminant proteins. The enrichment analysis performed using the tools GSEA, STRING and applying the Fisher’s exact test, revealed that the applied bacterial lysis protocol allowed the recovery of a highly enriched periplasmic fraction. A deep analysis of the identified proteins consented to evidence the expression and the localization of periplasmic proteins that to the best of our knowledge have never been reported in literature. The second part of my thesis was conducted in the frame of a larger project aimed at developing a platform for de-novo sequencing of antibodies raised after vaccination or infection. An initial experimental part of this project foresees the use of multiple enzymes to obtain as many peptides as possible from the enzymatic digestion of antibodies purified from vaccinated or infected donors. In my work, an innovative proteolytic enzyme, the prolyl endopeptidase (PSE), was tested to evaluate its suitability for peptide de-novo sequencing. The enzymatic activity of PSE was assessed at 37°C under different conditions to define the optimal pH and reaction time yielding the highest sequence coverage of a recombinant monoclonal antibody and to evaluate the site specificity. My results showed that PSE preferentially cleaves protein substrates at the C-term of proline. In addition, minor cleavage sites at residues serine, alanine, glycine and threonine were observed. Comparing the results obtained at different pH conditions and for distinct times of incubation, it was assessed that performing the incubation of the substrate with the enzyme in citric acid - disodium phosphate buffer pH 3 for 3 hours permits to reach the highest sequence coverage and the highest cleavage specificity, therefore in the following experiments these digestion parameters will be used to perform de-novo sequencing.
La mia tesi si è concentrata su due aspetti delle applicazioni di proteomica nel campo della vaccinologia che richiedono il supporto della bioinformatica. Il primo aspetto si riferisce alla gestione dei dati. Sono stati applicati metodi computazionali per lo studio di un protocollo innovativo che fa uso di tripsina in minime quantità per brevi tempi di digestione per accedere alle proteine periplasmatiche di Pseudomonas aeruginosa, senza causare la lisi dell'intero batterio, che potrebbe provocare la fuoriuscita di altre frazioni subcellulari. I tool CELLO e PSORTb sono stati utilizzati inizialmente per prevedere la localizzazione delle proteine identificate e quindi per discriminare la presenza di proteine contaminanti. L'analisi di arricchimento, eseguita applicando il test esatto di Fisher e i tool GSEA e STRING, ha rivelato che il protocollo di lisi batterica applicato ha permesso il recupero di una frazione periplasmatica altamente arricchita. Un'analisi approfondita delle proteine identificate ha permesso di evidenziare l'espressione e la localizzazione di proteine periplasmatiche che, al meglio delle nostre conoscenze, non sono mai state riportate in letteratura. La seconda parte della mia tesi è stata condotta nell'ambito di un progetto più ampio volto a sviluppare una piattaforma per il sequenziamento de-novo degli anticorpi sviluppati dopo la vaccinazione o l'infezione. Una prima parte sperimentale di questo progetto prevede l'utilizzo di più enzimi per ottenere il maggior numero possibile di peptidi dalla digestione enzimatica di anticorpi purificati da donatori vaccinati o infetti. Nel mio lavoro, un enzima proteolitico innovativo, la prolil endopeptidasi (PSE), è stato testato per valutare la sua idoneità al sequenziamento de-novo di proteine. L'attività enzimatica di PSE è stata valutata a 37°C in diverse condizioni per definire il pH ottimale e il tempo di incubazione che hanno prodotto il massimo sequence coverage di un anticorpo monoclonale ricombinante e per valutare la specificità del sito di taglio. I miei risultati hanno mostrato che PSE scinde preferenzialmente i substrati proteici al C-terminale di prolina. Inoltre, sono stati osservati siti di taglio minori ai residui di serina, alanina, glicina e treonina. Confrontando i risultati ottenuti a diverse condizioni di pH e per tempi distinti di incubazione, è stato valutato che l'esecuzione dell'incubazione del substrato con l'enzima nel buffer acido citrico - fosfato disodico pH 3 per 3 ore consente di raggiungere il massimo sequence coverage e la più alta specificità di taglio, pertanto tali condizioni sperimentali saranno utilizzati per eseguire il sequenziamento de-novo.
Computational methods applied to proteomics in vaccine discovery : from label-free quantification to de-novo protein sequencing
Della Corte, Irma
2020/2021
Abstract
My thesis focused on two aspects of proteomics applications in the vaccinology field that requires supportive bioinformatic applications. The first one refers to data management. Computational methods were applied for the identification of an innovative protocol making use of trypsin in sub-minute amount for short digestion times to access to the Pseudomonas aeruginosa periplasmic proteins, without causing the lysis of the whole bacteria, which could have provoked the leakage of other bacterial cellular fractions. The tools CELLO and PSORTb were first used to predict the localization of the identified proteins and therefore to discriminate the presence of contaminant proteins. The enrichment analysis performed using the tools GSEA, STRING and applying the Fisher’s exact test, revealed that the applied bacterial lysis protocol allowed the recovery of a highly enriched periplasmic fraction. A deep analysis of the identified proteins consented to evidence the expression and the localization of periplasmic proteins that to the best of our knowledge have never been reported in literature. The second part of my thesis was conducted in the frame of a larger project aimed at developing a platform for de-novo sequencing of antibodies raised after vaccination or infection. An initial experimental part of this project foresees the use of multiple enzymes to obtain as many peptides as possible from the enzymatic digestion of antibodies purified from vaccinated or infected donors. In my work, an innovative proteolytic enzyme, the prolyl endopeptidase (PSE), was tested to evaluate its suitability for peptide de-novo sequencing. The enzymatic activity of PSE was assessed at 37°C under different conditions to define the optimal pH and reaction time yielding the highest sequence coverage of a recombinant monoclonal antibody and to evaluate the site specificity. My results showed that PSE preferentially cleaves protein substrates at the C-term of proline. In addition, minor cleavage sites at residues serine, alanine, glycine and threonine were observed. Comparing the results obtained at different pH conditions and for distinct times of incubation, it was assessed that performing the incubation of the substrate with the enzyme in citric acid - disodium phosphate buffer pH 3 for 3 hours permits to reach the highest sequence coverage and the highest cleavage specificity, therefore in the following experiments these digestion parameters will be used to perform de-novo sequencing.| File | Dimensione | Formato | |
|---|---|---|---|
|
Tesi_IDC.pdf
non accessibile
Dimensione
2.84 MB
Formato
Adobe PDF
|
2.84 MB | Adobe PDF | Visualizza/Apri |
I documenti in POLITesi sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/10589/179954