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Combinatorial peptide ligand libraries for the analysis of low‐expression proteins: Validation for normal urine and definition of a first protein MAP
Author(s) -
Santucci Laura,
Candiano Giovanni,
Bruschi Maurizio,
D'Ambrosio Chiara,
Petretto Andrea,
Scaloni Andrea,
Urbani Andrea,
Righetti Pier G.,
Ghiggeri Gian M.
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100404
Subject(s) - urine , reproducibility , chromatography , peptide , ligand (biochemistry) , elution , chemistry , computational biology , bioinformatics , biology , biochemistry , receptor
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.