ITNA20110012A1 - INSULATION AND PURIFICATION PROCEDURE OF URINARY EXOSOMES FOR RESEARCH OF PROTEIN BIOMARCERS - Google Patents
INSULATION AND PURIFICATION PROCEDURE OF URINARY EXOSOMES FOR RESEARCH OF PROTEIN BIOMARCERS Download PDFInfo
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- ITNA20110012A1 ITNA20110012A1 IT000012A ITNA20110012A ITNA20110012A1 IT NA20110012 A1 ITNA20110012 A1 IT NA20110012A1 IT 000012 A IT000012 A IT 000012A IT NA20110012 A ITNA20110012 A IT NA20110012A IT NA20110012 A1 ITNA20110012 A1 IT NA20110012A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
Description
Descrizione dell’invenzione industriale avente il titolo “Procedura di isolamento e di purificazione degli esosomi urinari per la ricerca di biomarcatori proteici” Description of the industrial invention entitled "Procedure for isolation and purification of urinary exosomes for the search for protein biomarkers"
, ,
Gli esosomi sono coinvolti in una vasta gamma di eventi fisiologici come la modulazione del sistema immunitario, la comunicazione intercellulare e le funzioni paracrine, e di eventi patogenetici come tumore, traffico di patogeni virali e prioni. L’interesse scientifico-tecnologico per gli esosomi è in continuo aumento. ( 1 ) Exosomes are involved in a wide range of physiological events such as modulation of the immune system, intercellular communication and paracrine functions, and in pathogenetic events such as tumor, viral pathogen trafficking and prions. The scientific-technological interest in exosomes is constantly increasing. (1)
A.) Campo della tecnica a cui l’invenzione fa riferimento A.) Field of the technique to which the invention refers
La presente invenzione riguarda la procedura di isolamento e purificazione di esosomi urinari con lo scopo finale di produrre campioni di alta qualità con una resa riproducibile per la ricerca di biomarcatori proteici. Il campo specifico a cui l'invenzione fa riferimento è la biotecnologia. The present invention relates to the procedure of isolation and purification of urinary exosomes with the final aim of producing high quality samples with a reproducible yield for the search for protein biomarkers. The specific field to which the invention refers is biotechnology.
B.) Stato della tecnica preesistente B.) Pre-existing state of the art
Le procedure preesistenti di isolamento di esosomi da urina si basano su i.) centrifugazione differenziale (2), ii.) filtrazione su membrane (3, 4) e iii.) ultracentrifugazione in gradiente di saccarosio o su cuscino di saccarosio in ossido di deuterio (D20) (5) o sulla combinazione delle procedure sopra elencate con metodi di immunoaffinità. I campioni preparati secondo le procedure preesistenti sono caratterizzati da un’alta e variabile concentrazione d’uromodulina copurificata, fenomeno noto nella letteratura specialistica come "problema dell'uromodulina” (6). Recentemente è stato suggerito l'uso di ditiotreitolo (DTT) per la rimozione degli aggregati ad alto peso molecolare mediante riduzione dei ponti disolfuro intermolecolari dell’uromodulina (7). Il trattamento con DTT migliora la resa della preparazione di esosomi urinari ma non è in grado di risolvere in modo efficace il “problema deH’uromodulina". Per questo i campioni preparati con le procedure anteriori non sono adatti per l'analisi proteomica quantitativa. Pre-existing exosome isolation procedures from urine are based on i.) Differential centrifugation (2), ii.) Membrane filtration (3, 4) and iii.) Sucrose gradient ultracentrifugation or sucrose cushion in deuterium oxide (D20) (5) or on the combination of the procedures listed above with immunoaffinity methods. The samples prepared according to the pre-existing procedures are characterized by a high and variable concentration of copurified uromodulin, a phenomenon known in the specialized literature as the "uromodulin problem" (6). Recently it has been suggested the use of dithiothreitol (DTT) for the removal of high molecular weight aggregates by reducing the intermolecular disulfide bridges of uromodulin. (7) Treatment with DTT improves the yield of the preparation of urinary exosomes but is not able to effectively solve the "uromodulin problem" . Therefore, samples prepared with the previous procedures are not suitable for quantitative proteomics analysis.
Brevetti legati alla purificazione e all'uso di esosomi - ricerca di anteriorità Patents related to the purification and use of exosomes - prior art search
Per quanto a conoscenza degli inventori, attualmente esiste solo un brevetto (3) che rivendica la procedura di isolamento di vescicole di membrana da fluidi biologici tra cui le urine. Il procedimento brevettato (3) si basa sulla tecnica di microfiltrazione su membrana (4). A parte questo (3), esistono numerosi brevetti che rivendicano l’uso di esosomi da fluidi biologici per la diagnosi e la terapia (vedi sotto). Questi però applicano i procedimenti, descritti nella letteratura scientifica, per la purificazione degli esosomi da fluidi biologici (alcuni dei quali includono anche le urine), come la centrifugazione differenziale (8-12), l’ultracentrifugazione in gradiente di saccarosio e/o su cuscino di saccarosio (9), e/o la combinazione di questi con l'isolamento di specifici esosomi mediante immunoaffinità (9, 11, 13). I brevetti 9, 11 e 13 impiegano anticorpi specifici per l'immunopurificazione degli esosomi che presentano gli antigeni specifici per la malattia target. Da sottolineare, che la qualità e la purezza degli esosomi isolati con le procedure anteriori sono adatte per la caratterizzazione di micro-RNA esosomiali ma non per la caratterizzazione di proteine mediante analisi proteomica quantitativa finalizzata alla ricerca di biomarcatori proteici. To the best of the inventors' knowledge, there is currently only one patent (3) which claims the procedure for isolating membrane vesicles from biological fluids including urine. The patented process (3) is based on the membrane microfiltration technique (4). Apart from this (3), there are numerous patents that claim the use of exosomes from biological fluids for diagnosis and therapy (see below). However, these apply the procedures, described in the scientific literature, for the purification of exosomes from biological fluids (some of which also include urine), such as differential centrifugation (8-12), ultracentrifugation in sucrose gradient and / or sucrose cushion of sucrose (9), and / or the combination of these with the isolation of specific exosomes by immunoaffinity (9, 11, 13). Patents 9, 11 and 13 employ specific antibodies for the immunopurification of exosomes presenting the specific antigens for the target disease. It should be emphasized that the quality and purity of the exosomes isolated with the previous procedures are suitable for the characterization of exosomal micro-RNAs but not for the characterization of proteins by quantitative proteomic analysis aimed at the search for protein biomarkers.
Brevetti legati all'uso di esosomi nella terapia e nella diagnosi Patents linked to the use of exosomes in therapy and diagnosis
Sono stati utilizzati esosomi isolati da varie cellule come, per esempio, cellule dendritiche (DC), linfociti T e cellule tumorali, per sviluppare vaccini privi di cellule per l'immunoterapia del cancro. (14, 15) Recentemente sono state completate tre “clinical trials” in fase I e nuovi studi sottolineano le prospettive positive dell’applicazione degli esosomi nel trattamento del tumore. L'immunoterapia mediante esosomi è un campo molto innovativo, nonostante ciò sono stati già rilasciati alcuni brevetti (12, 16-19). Gli esosomi derivanti da cellule tumorali sono noti per essere una ricca fonte di antigeni legati al rigetto del tumore. (12) Alcuni brevetti rivendicano l'uso di esosomi naturali (12) o modificati (16, 19) derivanti da cellule tumorali. Esosomi privi di uno o più degli antigeni immunosoppressivi sono stati brevettati come vaccini nella cura di alcune malattie, tra cui il cancro (19). Inoltre, alcune proteine isolate da linee cellulare modificate e da siero che si legano selettivamente ai marcatori specifici degli esosomi (“exosome ligands”) sono state brevettate per l'uso terapeutico, profilattico o diagnostico. (16) Vescicole di membrana selettivamente funzionalizzate per esprimere un polipeptide da costrutti genetici e cellule ricombinanti sono state usate per produrre anticorpi che inducono o regolano la risposta immunitaria. L'utilizzo di lactadherina per esprimere selettivamente polipeptidi nelle vescicole di membrana è stato brevettato per l'uso terapeutico, profilattico e diagnostico (17). Exosomes isolated from various cells, such as dendritic cells (DCs), T lymphocytes and cancer cells, have been used to develop cell-free vaccines for cancer immunotherapy. (14, 15) Three "clinical trials" in phase I have recently been completed and new studies highlight the positive prospects of the application of exosomes in the treatment of cancer. Immunotherapy using exosomes is a very innovative field, despite this some patents have already been issued (12, 16-19). Exosomes derived from cancer cells are known to be a rich source of antigens related to tumor rejection. (12) Some patents claim the use of natural (12) or modified (16, 19) exosomes derived from tumor cells. Exosomes lacking one or more of the immunosuppressive antigens have been patented as vaccines in the treatment of certain diseases, including cancer (19). In addition, some proteins isolated from modified cell lines and from serum that selectively bind to specific markers of exosomes (“exosome ligands”) have been patented for therapeutic, prophylactic or diagnostic use. (16) Membrane vesicles selectively functionalized to express a polypeptide from genetic constructs and recombinant cells were used to produce antibodies that induce or regulate the immune response. The use of lactadherine to selectively express polypeptides in membrane vesicles has been patented for therapeutic, prophylactic and diagnostic use (17).
Ultimamente sono stati depositati un numero crescente di brevetti per sfruttare l'uso degli esosomi anche nella diagnosi medica. (9-13, 20, 21) Prevalentemente, essi rivendicano l’uso di microRNA da esosomi (13, 20, 21) e in misura minore di alcune proteine da esosomi (9, 11). La maggior parte di questi brevetti rivendica l'uso degli esosomi nella diagnosi del cancro (10, 12, 13, 21) e delle malattie infettive (10, 11, 20). E’ stato brevettato, per esempio, sia un metodo per la diagnosi di nefropatia associata all'infezione da HIV basato sulla rilevazione della presenza degli anticorpi specifici per HIV (11) che uno per l’epatite da virus HCV basato sulla rilevazione HCV RNAs (8, 20) negli esosomi. Recentemente l'uso degli esosomi è stato brevettato anche per la diagnosi prenatale. (9) Lately, an increasing number of patents have been filed to exploit the use of exosomes also in medical diagnosis. (9-13, 20, 21) Mostly, they claim the use of microRNAs from exosomes (13, 20, 21) and to a lesser extent some proteins from exosomes (9, 11). Most of these patents claim the use of exosomes in the diagnosis of cancer (10, 12, 13, 21) and infectious diseases (10, 11, 20). For example, both a method for the diagnosis of nephropathy associated with HIV infection based on the detection of the presence of specific antibodies for HIV (11) and one for hepatitis from HCV virus based on the detection of HCV RNAs ( 8, 20) in exosomes. Recently, the use of exosomes has also been patented for prenatal diagnosis. (9)
“ExoQuick” è una procedura semplice per isolare gli esosomi da campioni biologici utilizzando un tampone specifico sotto brevetto. ExoQuick è commercializzata dalla SS/, System Biosciences. La preparazione del campione è semplice è adatto per l'analisi di microRNA, ma dai dati pubblicati dal venditore, si evince che il metodo non è adatto per la produzione di esosomi per la ricerca di biomarcatori proteici mediante proteomica. “ExoQuick” is a simple procedure to isolate exosomes from biological samples using a specific patented buffer. ExoQuick is marketed by SS /, System Biosciences. The sample preparation is simple and suitable for microRNA analysis, but from the data published by the vendor, it appears that the method is not suitable for the production of exosomes for the search for protein biomarkers by proteomics.
“ExoTest” è un kit ELISA “double-sandwich" commercializzato da HansaBioMed (provvisoriamente brevettato negli Stati Uniti, US61/062, 528) che consente la rivelazione, quantificazione e caratterizzazione degli esosomi nel plasma umano basato sull'espressione di alcune proteine “housekeeping'' come CD63, Rab-5b e marcatori come la caveolina-1 associati al tumore. Il test è stato sviluppato principalmente per la diagnosi del cancro, ma dovrebbe essere esteso anche ad altre malattie. "ExoTest" is a "double-sandwich" ELISA kit marketed by HansaBioMed (provisionally patented in the United States, US61 / 062, 528) that allows the detection, quantification and characterization of exosomes in human plasma based on the expression of some "housekeeping" proteins '' such as CD63, Rab-5b and tumor-associated markers such as caveolin-1. The test was developed primarily for the diagnosis of cancer, but should be extended to other diseases as well.
C.) Esposizione dell’invenzione C.) Presentation of the invention
Rispetto al procedimento anteriore più efficace per la purificazione, ossia il procedimento iii.), l'invenzione ha tre nuove caratteristiche importanti: Compared to the more effective prior process for purification, i.e. process iii.), The invention has three new important features:
uso di un tampone adatto a tenere l’uromodulina in soluzione use of a buffer suitable for keeping uromodulin in solution
Per la solubilizzazione di aggregati d’uromodulina sono stati testati vari tamponi in diverse condizioni sperimentali. In accordo con studi precedenti eseguiti in vitro (22), abbiamo visto che per evitare la formazione di aggregati d’uromodulina durante la preparazione degli esosomi da urina è importante il pH alcalino, la bassa concentrazione di sodio e calcio e l'alta diluizione dei campioni. In particolare abbiamo scoperto che rispetto al tampone PBS composto da Na2HP0410 mM,. NaH2P042 mM e NaCI 150 mM a pH 7,2 che viene usato solitamente per la purificazione degli esosomi urinari, il tampone Tris-HCI (tris(idrossimetil)amminometano cloridrato) 10 mM a pH alcalino (pH 8,6) è ideale per mantenere l'uromodulina in soluzione e gli esosomi intatti. For the solubilization of uromodulin aggregates, various buffers were tested under different experimental conditions. In accordance with previous studies performed in vitro (22), we have seen that to avoid the formation of uromodulin aggregates during the preparation of exosomes from urine, the alkaline pH, the low concentration of sodium and calcium and the high dilution of the samples. In particular we found that compared to the PBS buffer composed of Na2HP0410 mM ,. NaH2P042 mM and NaCI 150 mM at pH 7.2 which is usually used for purification of urinary exosomes, the 10 mM Tris-HCI (tris (hydroxymethyl) aminomethane hydrochloride) buffer at alkaline pH (pH 8.6) is ideal for maintaining uromodulin in solution and intact exosomes.
• ultracentrifugazione su doppio cuscino di saccarosio in ossido di deuterio • ultracentrifugation on a double cushion of sucrose in deuterium oxide
Come secondo punto, è stata introdotta nella procedura di centrifugazione iterativa sequenziale (vedi dettagli sotto) l'ultracentrifugazione su doppio cuscino di saccarosio in D20 del campione solubilizzato nel tampone di cui sopra. Questo passaggio permette in modo semplice, senza l'uso del gradiente di saccarosio, di separare efficientemente gli esosomi dalle vescicole più grandi e/o frammenti di membrane più densi. As a second point, ultracentrifugation on a double cushion of sucrose in D20 of the sample solubilized in the above buffer was introduced in the sequential iterative centrifugation procedure (see details below). This step allows in a simple way, without the use of the sucrose gradient, to efficiently separate the exosomes from the larger vesicles and / or fragments of denser membranes.
■ compatibilità con metodi di proteomica ■ compatibility with proteomics methods
Gli esosomi purificati possono essere utilizzati per la ricerca di biomarcatori proteici mediante tecniche di proteomica come noto nell’arte. Per la proteomica quantitativa è fondamentale partire da un campione biologico puro e riproducibile. Le procedure anteriori di purificazione degli esosomi sono caratterizzate da una variabile ed alta concentrazione di uromodulina dovuta al fatto che l'uromodulina forma filamenti ed aggregati insolubili sia nell’urina che nel tampone utilizzato per la preparazione degli esosomi nell'arte anteriore. L’alta quantità di contaminazione, nella proteomica, rende difficile l'identificazione delle proteine in bassa concentrazione sia a causa dell'effetto della ‘soppressione del segnale' che dell’alterazione della concentrazione dinamica degli analiti. La variabilità della contaminazione da campione a campione invece rende l'analisi quantitativa difficilmente riproducibile. The purified exosomes can be used for the search for protein biomarkers using proteomics techniques as known in the art. For quantitative proteomics it is essential to start from a pure and reproducible biological sample. The prior exosome purification procedures are characterized by a variable and high concentration of uromodulin due to the fact that uromodulin forms insoluble filaments and aggregates both in urine and in the buffer used for the preparation of exosomes in the prior art. The high amount of contamination, in proteomics, makes it difficult to identify proteins in low concentrations both due to the effect of 'signal suppression' and the alteration of the dynamic concentration of the analytes. The variability of contamination from sample to sample, on the other hand, makes the quantitative analysis difficult to reproduce.
La procedura che sfrutta tre aspetti innovativi di cui sopra rispetto alle procedure anteriori può essere riassunta come segue: The procedure that exploits three innovative aspects mentioned above compared to the previous procedures can be summarized as follows:
1 . Raccolta e stoccaggio del campione urina - si raccolgono le seconde urine del mattino, si aggiunge ad esse una miscela d'inibitori di proteasi e fosfatasi e si conservano a -80 °C secondo le procedure pubblicate nella letteratura attinente (23). I campioni sono scongelati prima dell’utilizzo. 1. Urine sample collection and storage - second morning urine is collected, a mixture of protease and phosphatase inhibitors is added to it and stored at -80 ° C according to procedures published in the relevant literature (23). The samples are thawed before use.
2. Isolamento e purificazione degli esosomi da urina 2. Isolation and purification of exosomes from urine
a. Centrifugazione sequenziale: il campione è sottoposto a centrifugazione iterativa sequenziale: to. Sequential centrifugation: the sample is subjected to sequential iterative centrifugation:
400xg, 15 min, 4 °C; 800xg, 15 min, 4 °C; 15000xg, 15 min, 4 °C. Il surnatante è recuperato e ricentrifugato a 100000xg per 1 ora a 4 °C. Il pellet contenente gli esosomi è recuperato. b. Solubilizzazione differenziale'. Il pellet viene risospeso nel tampone di solubilizzazione, preferibilmente in tampone Tris 10 mM pH 8,6, ricentrifugato a 15000xg, 20 min, 4 °C gli esosomi urinari sono recuperati nel surnatante mentre il pellet contenente gli aggregati non solubili d’uromodulina è allontanato. 400xg, 15 min, 4 ° C; 800xg, 15 min, 4 ° C; 15000xg, 15 min, 4 ° C. The supernatant is recovered and re-centrifuged at 100000xg for 1 hour at 4 ° C. The pellet containing the exosomes is recovered. b. Differential solubilization '. The pellet is resuspended in the solubilization buffer, preferably in 10 mM Tris buffer pH 8.6, re-centrifuged at 15000xg, 20 min, 4 ° C the urinary exosomes are recovered in the supernatant while the pellet containing the insoluble aggregates of uromodulin is removed. .
c. Ultracentrifugazione su doppio cuscino (Figura 1): si prepara una soluzione di saccarosio 1 M e una di saccarosio 2 M entrambe in Tris 20 mM (pH 8.6) D20, queste sono poi stratificate una sopra l’altra nel tubo da ultracentrifuga sotto il campione in modo da formare due "cuscini” come noto nello stato dell'arte. Il campione è sottoposto a ultracentrifugazione a HOOOOxg, 3 ore a 4 °C. Le vescicole di membrana sono recuperate da entrambe le frazioni di saccarosio, diluite nel tampone di solubilizzazione e ultracentrifugati a HOOOOxg 90 min, a 4 °C per due volte e recuperate nel pellet. c. Ultracentrifugation on double pillow (Figure 1): a 1 M sucrose solution and a 2 M sucrose solution are prepared both in 20 mM Tris (pH 8.6) D20, these are then layered one on top of the other in the ultracentrifuge tube under the sample so as to form two "pillows" as known in the state of the art. The sample is subjected to ultracentrifugation at HOOOOxg, 3 hours at 4 ° C. The membrane vesicles are recovered from both sucrose fractions, diluted in the solubilization buffer and ultracentrifuged at HOOOOxg 90 min, at 4 ° C twice and recovered in the pellet.
D.) Esempio di attuazione dell’invenzione D.) Example of implementation of the invention
Il procedimento può essere applicato a volumi diversi di campioni di urina. Nell'esempio il campione è composto da un pool di urine di 200 mL raccolto da volontari maschi sani. La concentrazione delle proteine da esosomi isolate con la procedura oggetto dell'invenzione è di 0.1-0.125 pg/mL di urine determinata mediante procedure note (BCA, Bradford etc.) dopo la lisi delle vescicole. La resa più bassa rispetto alle procedure anteriori è dovuta alla rimozione del 50-70% di contaminazione da proteine d'urina abbondanti, che di solito si presentano in concentrazioni variabili nei campioni preparati con procedure tradizionali. The procedure can be applied to different volumes of urine samples. In the example, the sample is composed of a 200 mL urine pool collected from healthy male volunteers. The concentration of the proteins from exosomes isolated with the procedure object of the invention is 0.1-0.125 pg / mL of urine determined by known procedures (BCA, Bradford etc.) after the lysis of the vesicles. The lower yield compared to prior procedures is due to the removal of 50-70% of contamination from abundant urine proteins, which usually occur in varying concentrations in samples prepared with traditional procedures.
Il processo di isolamento/purificazione (punto 2) è stato ottimizzato, analizzando sia i pellets che i surnatanti ottenuti nelle fase di centrifugazione (punto 2a-2c), stimando la purezza e il contenuto di esosomi nei preparati. Per dimostrare che la qualità del campione è adeguata alla piattaforma per la ricerca di biomarcatori mediante proteomica, sono state effettuate le seguenti analisi: The isolation / purification process (point 2) has been optimized, analyzing both the pellets and the supernatants obtained in the centrifugation step (point 2a-2c), estimating the purity and the content of exosomes in the preparations. To demonstrate that the sample quality is adequate for the platform for biomarker research using proteomics, the following analyzes were performed:
1. Microscopia elettronica in trasmissione (TEM): L’analisi TEM degli esosomi è stata effettuata, come descritto nella letteratura (4). La TEM ha rivelato che il campione di esosomi urinari purificato con la nuova procedura è sostanzialmente omogeneo e l'immagine mostra vescicole di membrana tra i 30-80 nm di diametro. 1. Transmission electron microscopy (TEM): TEM analysis of the exosomes was performed, as described in the literature (4). TEM revealed that the urine exosome sample purified with the new procedure is substantially homogeneous and the image shows membrane vesicles between 30-80 nm in diameter.
2. Analisi SDS-PAGE: per ciascuna fase della procedura di purificazione sono stati analizzati 15 pg di proteine (sia sumatante che pellet) mediante 1 D SDS-PAGE in gradiente 4-12% e colorati con Coomassie blu colloidale. Il campione di partenza mostra il profilo SDS-PAGE caratteristico delle proteine da urine ed evidenzia in particolare la presenza di alte concentrazioni di uromodulina, albumina, pepsina, ecc. Dalla centrifugazione differenziale si ottiene un sumatante contenente le proteine solubili urinarie e una frazione grezza degli esosomi. La centrifugazione a bassa velocità del pellet risospeso utilizzando il tampone di solubilizzazione Tris, 10 mM rimuove una parte della contaminazione di uromodulina. La successiva ultracentrifugazione su doppio cuscino di saccarosio 1 M e saccarosio 2 M in D20 arricchisce in modo significativo la frazione saccarosio 1 M in esosomi (Figura 2A, lane 2). Rispetto alle procedure anteriori di centrifugazione differenziale (Figura 2A, lane 1), il nuovo procedimento produce un campione di esosomi urinari più puro. L’analisi SDS-PAGE delle proteine nella frazione di saccarosio 2 M (Figura 2B, lane 1) mostra un profilo caratteristico molto diverso da quello della frazione 1 M contenente gli esosomi (Figura 2B, lane 2). Alcune delle bande tagliate dalI’SDS-PAGE sono state analizzate mediante proteomica come noto nell'arte. L’identificazione di proteine quali per esempio la seminogelina, etc. indica che la frazione 2 M contiene vescicole chiamate prostasomi. Per mostrare l'eccellente riproducibilità del procedimento abbiamo ripetuto per cinque volte la procedura di isolamento/purificazione di esosomi, usando lo stessi pool di campioni di volontari sani. L’immagine dell’analisi SDS-PAGE mostra un pattern caratteristico e dimostra la riproducibilità della procedura. 2. SDS-PAGE analysis: for each step of the purification procedure 15 µg of proteins (both sumatant and pellet) were analyzed by 1 D SDS-PAGE in 4-12% gradient and stained with colloidal blue Coomassie. The starting sample shows the SDS-PAGE profile characteristic of urine proteins and highlights in particular the presence of high concentrations of uromodulin, albumin, pepsin, etc. By differential centrifugation, a sumatant is obtained containing the urinary soluble proteins and a crude fraction of the exosomes. Low speed centrifugation of the resuspended pellet using Tris solubilization buffer, 10 mM removes some of the uromodulin contamination. The subsequent ultracentrifugation on a double cushion of 1 M sucrose and 2 M sucrose in D20 significantly enriches the 1 M sucrose fraction in exosomes (Figure 2A, lane 2). Compared to prior differential centrifugation procedures (Figure 2A, lane 1), the new procedure produces a purer urine exosome sample. SDS-PAGE analysis of the proteins in the 2 M sucrose fraction (Figure 2B, lane 1) shows a very different characteristic profile from that of the 1 M fraction containing the exosomes (Figure 2B, lane 2). Some of the bands cut by the SDS-PAGE were analyzed by proteomics as known in the art. The identification of proteins such as semi-gelin, etc. indicates that the 2 M fraction contains vesicles called prostasomes. To show the excellent reproducibility of the procedure we repeated the exosome isolation / purification procedure five times, using the same pool of samples from healthy volunteers. The SDS-PAGE analysis image shows a characteristic pattern and demonstrates the reproducibility of the procedure.
3. Analisi proteomica: 100 pg di campioni di esosomi preparati secondo la procedura oggetto d'invenzione sono stati separati mediante elettroforesi 1 D SDS-PAGE (15x20 cm) in gradiente 5-20%. Dopo la corsa elettroforetica, la lane del gel è stata tagliata in 75 bande da 1,5 mm di altezza, le proteine sono state digerite con tripsina ed i peptidi triptici estratti da ciascuna banda sono stati analizzati mediante nano-HPLC-ESI-MS/MS, come noto nell’arte. (24) L’analisi proteomipa permette di identificare centinaia di proteine in entrambe le frazioni. 3. Proteomic analysis: 100 µg of exosome samples prepared according to the procedure object of the invention were separated by 1 D SDS-PAGE electrophoresis (15x20 cm) in a 5-20% gradient. After the electrophoretic run, the gel lane was cut into 75 1.5 mm high bands, the proteins were digested with trypsin and the tryptic peptides extracted from each band were analyzed by nano-HPLC-ESI-MS / MS, as known in the art. (24) Proteomipa analysis allows to identify hundreds of proteins in both fractions.
Inoltre, l’analisi proteomica MudPIT in-soluzione eseguita come noto nell’arte ha confermato l’alta qualità del campione. In addition, the in-solution MudPIT proteomic analysis performed as known in the art confirmed the high quality of the sample.
Dai risultati ottenuti si evince che la procedura oggetto d'invenzione rispetto a quelle anteriori migliora la qualità del campione (rimuovendo efficacemente le proteine abbondanti d’urina che spesso copurificano con gli esosomi), è riproducibile e consente di utilizzare il campione per l'analisi proteomica. The results obtained show that the procedure object of the invention, compared to the previous ones, improves the quality of the sample (effectively removing the abundant urine proteins that often copurify with the exosomes), is reproducible and allows the sample to be used for analysis. proteomics.
E.) Legenda delle figure E.) Legend of the figures
Figura 1. Preparazione del campione - Schema del passaggio di preparazione del doppio cuscino di saccarosio e successivo passaggio di ultracentrifugazione del campione. Figure 1. Sample preparation - Scheme of the preparation step of the double sucrose cushion and subsequent step of ultracentrifugation of the sample.
Figura 2 Analisi 1 D SDS-PAGE comparativa che mostra la differenza tra A) una procedura anteriore descritta nelle letteratura scientifica e quella oggetto di invenzione per l'isolamento e purificazione degli esosomi urinari. Lane 1- Esosomi urinari isolati (15 pg) con la procedura anteriore mediante centrifugazione differenziale ed ultracentrifugazione in tampone PBS pH 7.2. Lane 2-Esososmi urinari (15 pg) isolati/purificati con la procedura oggetto del brevetto mediante ultracentrifugazione su doppio cuscino e solubilizzazione differenziale nel tampone. Lane M-Proteine Standard (Invitrogen). B) vescicole recuperate dal cuscino di saccarosio 2 M in D20 (Lane 1 , prostesomi urinari) e dal cuscino di saccarosio 1 M in D20 (Lane 2, esosomi urinari). Lane M: Proteine standard (Invitrogen) Figure 2 Comparative 1 D SDS-PAGE analysis showing the difference between A) an earlier procedure described in the scientific literature and the one object of invention for the isolation and purification of urinary exosomes. Lane 1- Urinary exosomes isolated (15 pg) with the previous procedure by differential centrifugation and ultracentrifugation in PBS buffer pH 7.2. Lane 2-Urinary exososms (15 pg) isolated / purified with the procedure object of the patent by ultracentrifugation on double pillow and differential solubilization in the buffer. Lane M-Protein Standard (Invitrogen). B) vesicles recovered from the 2 M sucrose cushion in D20 (Lane 1, urinary prostesomes) and from the 1 M sucrose cushion in D20 (Lane 2, urinary exosomes). Lane M: Standard Protein (Invitrogen)
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| WO2001082958A2 (en) * | 2000-04-27 | 2001-11-08 | Anosys, Inc. | Methods of producing membrane vesicles |
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| WO2001082958A2 (en) * | 2000-04-27 | 2001-11-08 | Anosys, Inc. | Methods of producing membrane vesicles |
| WO2009027703A2 (en) * | 2007-08-29 | 2009-03-05 | University Court Of The University Of Edinburgh | Identifying organ damage |
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