Evaluation of isotope-coded protein labeling (ICPL) in the quantitative analysis of complex proteomes.
An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE "classical" approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.
Paradela
Alberto
A
Laboratorio de Proteómica, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. alberto.paradela@cnb.csic.es
Marcilla
Miguel
M
Navajas
Rosana
R
Ferreira
Laura
L
Ramos-Fernandez
Antonio
A
Fernández
Marisol
M
Mariscotti
Javier Fernando
JF
García-del Portillo
Francisco
F
Albar
Juan Pablo
JP
D002247:Carbon Isotopes / Q000737:chemistry; D019411:Clinical Laboratory Techniques; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D020543:Proteome