Abal, Miguel

Ballesteros, Carlos

Canals, Francesc

Colomé, Núria

Doll, Andreas

García, Marta

Mir, M. Carmen

Morote, Juan

Reventós, Jaume

Rigau, Marina

Repositorio Institucional de la Universidad de Córdoba

54 3527(Ï0,&$1Ò0(52)(%5(52DSSHDUDQFHRIFDQFHU$OWKRXJK WKHVSHFL¿FLW\ IRUthese responses is high, typically only 5-20% of pa-tients demonstrate a response to any given antigen, which has limited the usefulness of single antigen responses as biomarkers. The recent development of protein microarrays may offer an ideal tool for screening for immune response to tumor antigens. These arrays offer the advantage that hundreds to thousands of different proteins can be printed and screened simultaneously and only require a few microliters of serum per assay.Prof. LaBaer’s group (Harvard Institute of Pro-teomics) has developed a novel method for produ-cing protein microarrays called nucleic acid progra-mmable protein arrays (NAPPA) that avoids the need to express and purify the proteins by substituting the printing of cDNAs on the arrays, which are then transcribed and translated in situ as needed at the time of the assay. NAPPA has been used successfu-lly to map the pairwise protein interactions of the human DNA replication complex. Here, we propose adapting the NAPPA protein microarray technology IRUXVHLQWKHUDSLGDQGHI¿FLHQWVFUHHQLQJRIVHUDfrom cancer patients for antibodies to 1000 known and potential tumor antigens in a multiplex format in order to better characterize the immune response to known tumor antigens, identify new informative tumor antigens and evaluate the value of using pat-terns of tumor antigen immune responses as biomar-kers in Colorectal Cancer.  For the validation of the possible biomarkers found in 20 different patients SUH	SRVWFKHPRWKHUDS\FXUUHQWO\ZHDUHXVLQJiFISH and SNPs approaches with the main goal to correlate genomics and functional proteomics. Introduction3URVWDWHVSHFL¿FDQWLJHQ36$VHUXPPHDVXUH-ment in combination with a digital rectal examinati-on (DRE) and transrectal ultrasound-guided biopsy (TURS) is currently the gold standard for prostate cancer (PCa) screening in Europe [1]. Nevertheless, 36$DQG'5(ODFNVLJQL¿FDQWVSHFL¿FLW\DQGELRSV\lacks ideal sensitivity (12-30% false negatives) [2, 3]. Therefore additional biomarkers are needed to supplement or potentially replace the currently used diagnostic techniques. As the secreted products from both, normal prostate epithelial cells as well as PCa cells, can be detected in the urine of men, their use as DSUR[LPDOERG\ÀXLGWRGHWHFW3&DLVYHU\DWWUDFWLYHsince they could be the best compromise between a minimal invasive technique accepted by a wider range of the male population and the possibility to obtain enough cells for a correct diagnosis [4, 5]. Objective:HVRXJKWWRGHWHUPLQHDSURWHRPLFSUR¿OHLQurine able to distinguish between the presence and absence of PCa.0DWHULDO	0HWKRGV:HXVHGDFRPELQDWLRQRISURWHRPLF WHFKQRO-ogies in aged matched post-Digital Rectal Exam (DRE) urine supernatants specimens to identify dif-ferentially expressed proteins in patients with PCa. )LUVWO\ZHGHSOHWHGKLVWRORJLFDO FRQ¿UPHG3&Durine samples and 9 control samples (age-matched patients with the typical background of benign pros-WDWHKLSHUSODV\%3+DWURSK\DQGFKURQLFLQÀDP-mation) using ProteoMiner (BioRad), a novel deple-tion technique that reduces the level of the most abundant species, while strongly concentrating the more dilute and rare ones. Then, Two-dimensional Detection of Prostate Cancer by Urine ProteomicsMarina Rigau1, Núria Colome3, Juan Morote2, Mª Carme Mir2, Carlos Ballesteros2, Marta Garcia1,Miguel Abal1, Francesc Canals3, Jaume Reventós1, Andreas Doll11Unitat de Recerca Biomèdica, Institut de Recerca. 2Servei d’Urologia. 3Laboratori de Proteòmica, Institut de Recerca. Hospital Vall d’Hebron, Barcelona3527(Ï0,&$1Ò0(52)(%5(52 55gel-based proteomic approach (2D-DIGE) coupled with matrix assisted laser desorption/ionization ti-PHRIÀLJKWPDVVVSHFWURPHWU\0$/',72)06and database mining experiment were performed to identify novel biomarkers of PCa in urine. Finally, we validated the candiadate biomarkers using Multi-ple Reaction Monitoring (MRM)-based assays, con-ducted by Liquid chromatography in conjunction with triple quadrupole mass spectrometry (LC-MS/MS) in a bigger cohort of urine samples.Results:HLGHQWL¿HGDSURWHRPLFSUR¿OHRISRWHQ-tial biomarkers (16 down and 10 up-expressed) for the detection of PCa in urine. Ingenuity pat-hways analysis (IPA) showed that the majority of these proteins are secreted components of several ZHOONQRZQIXQFWLRQDORIFDQFHUDQGLQÀDPPDWL-RQQHWZRUNV OLNH1)Ʉ%3'*)%ȕDQG36$:Hperformed MRM-based assays for 15 candidate biomarkers to quantify these in a sample set of 50 urine supernatants (Figure 1).Figure 1. 3URWHLQ LGHQWL¿FDWLRQE\SURWHRPLF FRPSDUDWLYH DQDO\VLV $3URWHR0LQHUGHSOHWLRQ WHFKQLTXHRYHUYLHZ %2D-DIGE experiment design, comparison of  9 urine PCa samples against 9 urine control samples. (C) Example of a representive gel of the DIGE experiment. (D) Example of up-expressed spot in PCa samples. (E) IPA network interaction RIRYHUH[SUHVVHGSURWHLQVSRWVUHG¿JXUHVDQGXQGHUH[SUHVVHGSURWHLQVSRWVJUHHQ¿JXUHV)050WUDQVLWLRQVRIDWU\SWLFSHSWLGHGHULYHGIURPRQHRIWKHFDQGLGDWHELRPDUNHUSURWHLQV*050EDVHGDEVROXWHTXDQWL¿FDWLRQRIDSHSWLGHabundance using a labeled synthetic peptide as internal standard.ConclusionsThese data demonstrate the ability of proteo-mic analyses to reveal novel biomarkers for PCa in urine, an important step forward in advancing accurate diagnosis which is currently the bottleneck for the ability to cure patients from PCa. MRM is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by LWV XQLTXH SRWHQWLDO IRU UHOLDEOH TXDQWL¿FDWLRQ RIanalytes of low abundance in complex mixtures, such urine samples[6]. These biomarkers could be used to develop a a simple diagnostic test (probably similar to the ELISA, either multiplex or strips, reactive to the 4-5 molecules most representative of WKHGLIIHUHQWLDOSUR¿OHWREHXVHGLQWKHKRVSLWDODQGoutpatient routines.References>@ &DWDORQD:-6PLWK'65DWOLII7/'RGGV.0HWDO0HDVXUHPHQWRISURVWDWHVSHFL¿Fantigen in serum as a screening test for prostate cancer. N Engl J Med 1991; 324: 1156-1161.56 3527(Ï0,&$1Ò0(52)(%5(52[2] Cervera Deval, J., Morales Olaya, F. J., Jornet Fayos, J., Gonzalez Anon, M., [Diagnostic value of the second prostate biopsies in males of risk. 6WXG\VWUDWL¿HGE\YDOXHRI36$@$FWDV8UROEsp 2004; 28: 666-671.[3] Raber, M., Scattoni, V., Salonia, A., Consonni, P., Rigatti, P., [Repeated ultrasound-guided transrectal prostate biopsy in patients with negative histologic test]. Arch Ital Urol Androl 2000; 72: 197-199.[4] Okamoto, A., Yamamoto, H., Imai, A., Ha-takeyama, S., et al., Protein profiling of post-prostatic massage urine specimens by surface-enhanced laser desorption/ionization WLPHRIÀLJKWPDVVVSHFWURPHWU\WRGLVFULPLQD-te between prostate cancer and benign lesions. Oncol Rep 2009; 21: 73-79.[5] M’Koma, A. E., Blum, D. L., Norris, J. L., Ko-yama, T., et al., Detection of pre-neoplastic and QHRSODVWLFSURVWDWHGLVHDVHE\0$/',SUR¿OLQJof urine. Biochem Biophys Res Commun 2007; 353: 829-834.[6] Lange, V., Picotti, P., Domon, B., Aebersold, R., Selected reaction monitoring for quanti-tative proteomics: a tutorial. Mol Syst Biol 2008; 4: 222.Introduction Osteoarthritis (OA) is the most common rheu-matic pathology, characterized mainly by cartilage degradation [1]. Despite its high prevalence, the diagnosis methods currently available are limited and lacked of sensitivity. Therefore, there is a con-siderable interest pointed in the characterization of QHZVSHFL¿F2$ELRPDUNHUVLQELRORJLFDOÀXLGV,Qthis work we set up a nLC-MALDI-MS method for OA biomarker search in complex mixtures, with the aim of obtaining a standardized protocol for serum DQGV\QRYLDOÀXLG6)SURWHLQSUR¿OLQJMethodsa) Sample preparation and immunodepletion:Serum and SF samples were obtained from OA pa-tients and control donors. Prior to protein depletion, SF was treated with hyaluronidase. The 20 most abun-NanoLC/mass spectrometry-based proteomic analysis of serum and synovial ÀXLGVDPSOHVIURPRVWHRDUWKULWLVSDWLHQWVPatricia Fernández-Puente, Jesús Mateos, Carolina Fernández-Costa, Cristina Ruiz-Romero, Francisco J BlancoOsteoarticular and Aging Research Lab, Nodo Asociado de Proteo-Red. INIBIC-Hospitalario Universitario A Coruña, Xubias 84, 15006 - A Coruña, SPAINdant proteins were removed from crude serum and SF using the Immunodepletion column ProteoPrep® 20 (Figure 1), according to manufacturer’s instructions (Sigma Aldrich). Depletion of abundant proteins was checked by SDS-PAGE separation of the proteins and LGHQWL¿FDWLRQ LQD0$/',72)72)V\VWHP(ABI). For both serum and SF depleted samples, pro-tein concentration was determined using a nanoDrop LQVWUXPHQW)LVKHU7KHUPR6FLHQWL¿F86$b) Pre-fractioning of the samples: For serum sam-ples, digestion of the depleted fractions was done with trypsin, and the peptides obtained were frac-tionated by strong cation exchange (SCX) liquid chromatography in a HP 1200 system (Agilent), using a  PolySulfoethyl column (PolyLC). SF pro-teins were separated by SDS-PAGE, using 10% acrylamide gels. The gel lanes were divided into 12 sections, excised and proteins were in-gel digested with trypsin following standard procedures. 

Detection of Prostate Cancer by Urine Proteomics

Detection of Prostate Cancer by Urine Proteomics

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Repositorio Institucional de la Universidad de Córdoba