title: Combinatorial peptide ligand libraries for the analysis of low-expression proteins: Validation for normal urine and definition of a first protein MAP.
abstract: In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorial peptide ligand library (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are (i) definition of the urine sample requirements, (ii) optimization of the urine/ligand ratio, (iii) essay conditions, (iv) en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter and intraessay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC-MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly developed studies on human diseases.
authors.affiliation: G Gaslini Children Hospital, Laboratory of Pathophysiology of Uremia, Genoa, Italy. gmarcoghiggeri@ospedale-gaslini.ge.it
authors.forename: Laura
authors.initials: L
authors.lastname: Santucci
authors.forename: Giovanni
authors.initials: G
authors.lastname: Candiano
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Chiara
authors.initials: C
authors.lastname: D'Ambrosio
authors.forename: Andrea
authors.initials: A
authors.lastname: Petretto
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Andrea
authors.initials: A
authors.lastname: Urbani
authors.forename: Pier G
authors.initials: PG
authors.lastname: Righetti
authors.forename: Gian M
authors.initials: GM
authors.lastname: Ghiggeri
mesh_terms: D020650:Combinatorial Chemistry Techniques; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D016482:Urinalysis / Q000379:methods*; D014556:Urine / Q000737:chemistry*
chemical_list: D008024:Ligands; D019151:Peptide Library; D011506:Proteins