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.     

Current trends in quantitative proteomics

It was inevitable that as soon as mass spectrometrists were able to tell biologists which proteins were in their samples, the next question would be how much of these proteins were present. This has turned out to be a much more challenging question. In this review, we describe the multiple ways that mass spectrometry has attempted to address this issue, both for relative quantitation and for absolute quantitation of proteins. There is no single method that will work for every problem or for every sample. What we present here is a variety of techniques, with guidelines that we hope will assist the researcher in selecting the most appropriate technique for the particular biological problem that needs to be addressed. We need to emphasize that this is a very active area of proteomics research—new quantitative methods are continuously being introduced and some ‘pitfalls’ of older methods are just being discovered. However, even though there is no perfect technique—and a better technique may be developed tomorrow—valuable information on biomarkers and pathways can be obtained using these currently available methods Copyright © 2009 John Wiley & Sons, Ltd.