Experimental Details - HuPA_00692
Experiment type Mass spectrometry
Short description Discovery of biomarkers for aggressive Bladder Cancer
Experimental description In this study a fractionation strategy of urinary proteins was applied based on the use of immobilized metal affinity chromatography (Ni2+-IMAC) for the discovery of biomarkers for aggressive Bladder Cancer. Urine samples from patients with non invasive (2 pools) and invasive (2 pools) BC were subjected to IMAC fractionation and eluted proteins were analyzed by 1D-SDS PAGE. Protein bands were excised, destained and in gel digestion was performed. Tryptic peptides were then analyzed by C18 reverse phase LC-MS/MS (Agilent 6330 – Ion trap). Raw data were loaded into Mascot Distiller 2.3.2 software and peaklists were generated. The peaklists were exported in mgf format and submitted to Mascot Server search engine. Protein searches were performed against SwissProt database (Uniprot_sprot, 2010_01) for human taxonomy, calculating 1 maximum miss cleavage for trypsin, oxidation of methionine and deamidation of asparagine or glycine as random modifications. Error tolerances were 1.5 Da and 0.7 Da for precursor and fragment mode respectively. Mascot result files were submitted to Scaffold 3 software (Proteome Science) for validation and meta analysis. For the evaluation of protein hits, a threshold confidence level (according to Scaffold) of 90% was selected with a minimum of 2 positive peptides per identification. False discovery rate for proteins was 1.2% as calculated automatically by the Scaffold software.
Principal Investigator's NameAntonia Vlahou
AddressBiomedical Research Foundation
Soranou Efesiou 4
Athens, Attiki 115 27
Data submitted byPanagiotis Zerefos
TitleM.Sc. Ph.D.
Published/Unpublished Unpublished
Journal name Not applicable
PubMed ID Not applicable
Sample source
Tissue: Urine [eVOC:EV:0000002]
Cell line:
Source organism Homo sapiens [Taxonomy:9606]
Labeling technique No
Protease used Trypsin
Is the sample from in gel Yes
Reduction and Alkylation No
Mass spectrometer
Instrument: Ion Trap 6330
Vendor: Agilent Technologies
Ionization method ESI
Fragmentation method CID
Mass tolerance used for database searching (MS) 1.5 Da
Mass tolerance used for database searching (MS/MS) 0.7 Da
Database used for searching Swissprot
Search engine used Mascot
Download MS raw dataset  
Download processed files  

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