Talanta 80 (2010) 1496–1502 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Evaluation of isotope-coded protein labeling (ICPL) in the quantitative analysis of complex proteomes Alberto Paradela a,∗ , Miguel Marcilla a , Rosana Navajas a , Laura Ferreira c , Antonio Ramos-Fernandez a , Marisol Fernández a , Javier Fernando Mariscotti b , Francisco García-del Portillo b , Juan Pablo Albar a a Laboratorio de Proteómica, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain b Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain c Unidad de Investigación, Hospital Universitario, Salamanca, Spain article info Article history: Available online 9 July 2009 Keywords: Stable isotopic labeling Mass spectrometry Quantitative proteomics ICPL abstract 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. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The development of new quantitative tools for the analysis and comparison of whole proteomes (differential proteomics) has been continuous during the past years. Traditional approaches, based on previous fractionation of complex proteomes by 2D-gel elec- trophoresis and comparison of the gel images generated thereby, are being complemented by new techniques that commonly make use of isotopic labeling of the samples to be compared, followed by liquid chromatography coupled to electrospray tandem mass spec- trometry [1,2]. Isotopic labeling can be performed in vivo (e.g., SILAC [3]), chemically (e.g., ICAT [4], cICAT [5], iTRAQ [6], Lys-tag [7], ICPL [8], TMT [9]) or enzymatically (e.g., 16 O/ 18 O [10,11]). In addition, label-free techniques show increasing acceptance as ultra-high per- formance liquid chromatography systems are becoming available and software developments required to compare multiple LC–MS runs are gaining efficiency and reliability [12]. Abbreviations: CID, collision-induced dissociation; 2D-PAGE, bidimensional polyacrilamide gel electrophoresis; ICAT, isotope-coded affinity tag; ICPL, isotope- coded protein labeling; iTRAQ, isotope tagging for relative and absolute quantification; LC ESI-MS/MS, liquid chromatography coupled to electrospray tan- dem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; ORF, open-reading frame; SILAC, stable isotope labeling with amino acids in cell culture. ∗ Corresponding author. E-mail address: alberto.paradela@cnb.csic.es (A. Paradela). Among differential proteomics methods based on stable isotopic labeling, ICPL is a non-isobaric technique devised to efficiently label primary amines profusely found in proteins. Relative quantifica- tion is achieved by comparison of the relative abundance (regarded as either peak intensity or area) of the differentially labeled pep- tides. The latest version of this reagent allows the comparison of up to four samples at the same time. Theoretically, ICPL over- comes some of the disadvantages found in other chemical labeling techniques, such as iTRAQ (poor detectability of the reporter ions in several types of mass spectrometers) or ICAT (low number of potentially labelable residues). However, the number of articles published to date using ICPL is noticeably low [13–15] when com- pared with other methods and no clear consensus exists on its feasibility as a quantitative tool. Here we describe a comprehen- sive evaluation of this technique using two different biological models. First, the proteome of the bacteriophage T4 capsid was labeled with the light or heavy version of ICPL and mixed in a 1:1 ratio. This sample was then used to evaluate the viability and robustness of the ICPL approach as well as to determine the opti- mal experimental parameters. In the second case, we compared a more complex sample composed of whole proteomes from a Salmonella enterica serovar Typhimurium virulent strain and an isogenic strain harboring a mutation in the Intracellular-growth- attenuator-A (IgaA1). The Intracellular-growth-attenuator-A (IgaA) is a S. enterica membrane protein that inhibits the RcsC-YojN-RcsB regulatory system preventing its over activation. Mutations in IgaA have been shown to drastically alter the bacterial transcriptome 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.06.083