Combinatorial peptide ligand libraries for the analysis of low-expression proteins: Validation for normal urine and definition of a first protein MAP

Laura Santucci, Giovanni Candiano, Maurizio Bruschi, Chiara D'Ambrosio, Andrea Petretto, Andrea Scaloni, Andrea Urbani, Pier G. Righetti, Gian M. Ghiggeri

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)509-515
Number of pages7
JournalProteomics
Volume12
Issue number4-5
DOIs
Publication statusPublished - Feb 2012

Keywords

  • 2-DE
  • Proteome maps
  • Reproducibility
  • Technology

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

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