Differential proteomic analysis using isotope‐coded protein‐labeling strategies: Comparison, improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34

Among differential proteomic methods based on stable isotopic labeling, isotope‐coded protein labeling (ICPL) is a recent non‐isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical‐labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post‐digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom–up approach). Optimization and validation of this Post‐digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems – a 2‐D clinorotation and 3‐D random positioning device – were used and the results were compared and discussed.