One of the most important and still not well investigated post-translational modification (PTMs) is proteolysis. Indeed, proteolytic events are involved with the protein degradation machinery and play a crucial role in cellular signalling and protein translocation. Proteolysis is also involved in the regulation of molecular mechanisms such as homeostasis or inflammation, being of ultimate importance in both cell life and death. Proteolytic events lead to the formation of neo N-termini, thus novel methods such as Terminal Amine Isotopic Labeling of Substrates (TAILS), Combined Fractional Diagonal Chromatography (COFRADIC) and Charge-based Fractional Diagonal Chromatography (ChaFRADIC) have been developed. Together with state-of-the-art-LC-MS we can perform in depth qualitative and quantitative studies of proteolytic events in the cell. Thus, it may provide a better understanding of molecular mechanisms associated with fisio-phatological pathways in order to develop novel diagnosis or therapeutic strategies. In particular, ChaFRADIC is an HPLC based method, whiled enrichment technique, which specifically captures free N-terminal peptides, depleting the non-modified peptides. In the first step free N-terminal peptides must be blocked with a tag, afterwards the sample is subjected to a first dimension in a Strong cation exchange chromatography using an optimized gradient, thus 5 fractions corresponding to the different charge state of the peptides are collected. In order to deplete non-modified peptides generated from the in vitro digestion a heavy acetylation reaction is performed and each collected fraction is subjected to re-chromatography. Thus, heavy acetylated peptide shift to an early retention time, while the N-termini of interest are retained in their corresponding retention time. In this thesis, it is reported an optimization step of ChaFRADIC technique introduced by Venne AS et al in 2013 [1], changing the primary amines labelling procedures with iTRAQ (Isobaric Tag for Relative and Absolute Quantification). The advandage is, also, attributed to the possibility to analyse until 8 different samples/conditions in one single experiment. Furthermore, three different enzymes (trypsin, GluC and subtilisin) have been used in the digestion step, in order to increase the accessibility of free and endogenously acetylated N-terminal peptides in order to study proteolytic evensts in Arabidopsis thaliana. Using this procedure it has been possible to quantify 2791 unique N-terminal peptides corresponding to 2249 different unique N-termini from 1270 Arabidopsis proteins. These data demonstrate the power, reproducibility and sensitivity of this method. Furthermore, these data allow to study and to discriminate between stabilizing/destabilizing functions of N-terminal amino acid residues of different proteins or isoforms in Arabidopsis in accordance with the N-end rule degradation pathway (or NERP). [1]A. Saskia Venne, F. Nora Vogtle, Chris Meisinger, Albert Sickmann and Rene P. Zahedi. Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N-Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase icp55. J. Proteome 2013, 12, 3823-3830.
Tra le modificazioni post traduzionali cui una proteina può andare incontro sicuramente la proteolisi rappresenta una tra le più importanti. Tuttavia ad oggi non esistono molti studi approfonditi in questo ambito. Le reazioni di proteolisi sono coinvolte in numerosi processi biochimici tra cui il catabolismo proteico, nei processi di cascata di trasduzione del segnale, nel trasporto alla destinazione finale delle proteine; la proteolisi inoltre è coinvolta nella regolazione di meccanismi cellulari quali l’omeostasi ed è cruciale nei processi processi infiammatori così come anche nei processi che regolano la vita e la morte cellulare. Per poter caratterizzare queste modificazioni post traduzionali, risulta fondamentale avere a disposizione delle tecniche analitiche. A tal proposito sono stati sviluppati diversi metodi come Terminal Amine Isotopic Labeling of Substrates (TAILS), Combined Fractional Diagonal Chromatography (COFRADIC) e Charge-based Fractional Diagonal Chromatography (ChaFRADIC) che permettono di separare e marcare selettivamente le proteine in combinazione a tecniche di spettrometria di massa. Questi approci sono inoltre molto indicati per capire i meccanismi molecolari associati a processi fisio-patologici ma anche per la progettazione di nuovi metodi diagnostici e terapeutici. In questo lavoro sono riportati i dati riguardanti l’ottimizzazione del ChaFRADIC (Venne AS et al in 2013 [1]) che ha come obbiettivo quello di dare la possibilità di caratterizzare i frammenti proteolitici presenti in un campione complesso . Questa metodologia prevede diversi passaggi, in primo luogo tutte le proteine, appartenenti ad una miscela complessa, vengono marcate chimicamente a livello delle ammine primarie, successivamente vengono digerite da uno più enzimi proteolitici e i peptidi ottenuti sono poi separati mediante cromatografia a scambio ionico. In seguito le frazioni ottenute vengono sottoposte ad una reazione di acetilazione sugli N-terminali neoformati e sottoposte nuovamente a cromatografia a scambio ionico accoppiata a spettrometria di massa. Le reazioni di marcatura sono una fase critica di questo processo e sono state ottimizzate grazie all’utilizzo di un sistema iTRAQ (Isobaric Tag for Relative and Absolute Quantification), questo permette anche di analizzare fino a 8 campioni contemporaneamente. Per studiare l’efficacia di questo metodo si sono utilizzate le proteine estratte da Arabidopsis Thaliana. Con questa procedura è stato possibile identificare 2249 differenti sequenze N-terminali provenienti da 1270 proteine di Arabidopsis Thaliana. I risultati ottenuti dimostrano il potere e la sensibilità di questo metodo. Questi dati hanno inoltre permesso di proporre il ruolo dell’amminoacido N-terminale nella stabilizzazione o nella destabilizzazione di differenti proteine o isoforme. (N-end rule degradation pathway or NERP). [1]A. Saskia Venne, F. Nora Vogtle, Chris Meisinger, Albert Sickmann and Rene P. Zahedi. Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N-Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase icp55. J. Proteome 2013, 12, 3823-3830.
An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana
PARETI, TOMMASO
2014/2015
Abstract
One of the most important and still not well investigated post-translational modification (PTMs) is proteolysis. Indeed, proteolytic events are involved with the protein degradation machinery and play a crucial role in cellular signalling and protein translocation. Proteolysis is also involved in the regulation of molecular mechanisms such as homeostasis or inflammation, being of ultimate importance in both cell life and death. Proteolytic events lead to the formation of neo N-termini, thus novel methods such as Terminal Amine Isotopic Labeling of Substrates (TAILS), Combined Fractional Diagonal Chromatography (COFRADIC) and Charge-based Fractional Diagonal Chromatography (ChaFRADIC) have been developed. Together with state-of-the-art-LC-MS we can perform in depth qualitative and quantitative studies of proteolytic events in the cell. Thus, it may provide a better understanding of molecular mechanisms associated with fisio-phatological pathways in order to develop novel diagnosis or therapeutic strategies. In particular, ChaFRADIC is an HPLC based method, whiled enrichment technique, which specifically captures free N-terminal peptides, depleting the non-modified peptides. In the first step free N-terminal peptides must be blocked with a tag, afterwards the sample is subjected to a first dimension in a Strong cation exchange chromatography using an optimized gradient, thus 5 fractions corresponding to the different charge state of the peptides are collected. In order to deplete non-modified peptides generated from the in vitro digestion a heavy acetylation reaction is performed and each collected fraction is subjected to re-chromatography. Thus, heavy acetylated peptide shift to an early retention time, while the N-termini of interest are retained in their corresponding retention time. In this thesis, it is reported an optimization step of ChaFRADIC technique introduced by Venne AS et al in 2013 [1], changing the primary amines labelling procedures with iTRAQ (Isobaric Tag for Relative and Absolute Quantification). The advandage is, also, attributed to the possibility to analyse until 8 different samples/conditions in one single experiment. Furthermore, three different enzymes (trypsin, GluC and subtilisin) have been used in the digestion step, in order to increase the accessibility of free and endogenously acetylated N-terminal peptides in order to study proteolytic evensts in Arabidopsis thaliana. Using this procedure it has been possible to quantify 2791 unique N-terminal peptides corresponding to 2249 different unique N-termini from 1270 Arabidopsis proteins. These data demonstrate the power, reproducibility and sensitivity of this method. Furthermore, these data allow to study and to discriminate between stabilizing/destabilizing functions of N-terminal amino acid residues of different proteins or isoforms in Arabidopsis in accordance with the N-end rule degradation pathway (or NERP). [1]A. Saskia Venne, F. Nora Vogtle, Chris Meisinger, Albert Sickmann and Rene P. Zahedi. Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N-Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase icp55. J. Proteome 2013, 12, 3823-3830.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/25240