Book Chapter

Analysis of Isotopic Labelling Vs. Label-Free Approaches for Quantitative Proteomics.


Baptiste L, Houyoux N, Matallana-Surget S & Wattiez R (2012) Analysis of Isotopic Labelling Vs. Label-Free Approaches for Quantitative Proteomics.. In: Leung H, Man T & Flores R (eds.) Integrative Proteomics. Croatia: InTech, pp. 327-346.;

For more than three decades, proteomics have been a crucial tool for deciphering the intricate molecular systems governing biology. O'Farrel was the first to utilise 2 dimensional gel electrophoresis (2DE) to perform actual complex proteomic analyses (O'Farrell, 1975). 2DE has very quickly emerged at the forefront of this rapidly growing field of research and has allowed for thousands of studies in widely varied domains. The development of 2D Fluorescence Gel Electrophoresis (2D DIGE) has provided more accurate and reliable proteins quantification due to the simultaneous migration on a same gel of samples to be compared, avoiding gel-to-gel variation. More recently, technological improvements in liquid chromatography and mass spectrometry have made it possible to develop so called "gel-free proteomics" in which, after total proteome enzymatic digestion, the produced peptides are separated with a high resolution chromatographic system and identified using tandem mass spectrometry. A gel-free approach presents a number of advantages over 2DE, such as a higher sensitivity, an easier automation of procedures to provide a better reproducibility and a reduced influence of intrinsic protein characteristics (pI, molecular weight, etc.). Nevertheless, a high complementarity between 2DE and gel-free approaches has been extensively reported (Finamore et al., 2010; Charro et al., 2011; Matallana-Surget et al., submitted), which suggests that both methods will continue to be considered together for a long time. Furthermore, 2DE also presents some advantages over a gel-free workflow approach in particular contexts. Indeed, 2DE presents the important benefit of allowing for the detection of protein isoforms, which is still complicated using gel-free approaches. Another example is an immunoproteomic workflow in which 2DE is followed by immunodetection, which allows for the targeted analysis and detection of antigen candidates.  An additional important difference between gel-free approaches and gel-based workflow is found in the quality of the quantitative data obtained. Indeed, 2DE quantification relies on spot volume measurement, which implies each protein is quantified based on a single data point. In contrast, multiple peptides from the same protein can be used for quantification using gel-free approaches. This major difference clearly indicates that gel-free workflowderived quantitative data are more statistically robust. However, before gel-free workflow approaches can be used in differential proteomics, the intrinsic limitation of mass spectrometry-based peptide analyses, the ion suppression effect, must first be addressed.

Publication date31/12/2012
Publisher URL
Place of publicationCroatia