5.4. Differential proteomics of Paenibacillus larvae
Liquid chromatography coupled directly to mass spectrometry provides another means for monitoring a proteome or changes in a proteome. Such global monitoring of changes in levels of proteins in response to a stimulus (e.g. a pathogen such as P. larvae) can provide very direct insight into the molecular mechanisms employed to respond to that challenge. For example, bee larvae up-regulate expression and activation of phenoloxidase in response to a P. larvae challenge (Chan et al., 2009). Mass spectrometry is currently the favoured detection method for monitoring the entire protein component of a system (i.e. the proteome), and various methods exist for comparing protein expression in one state to that in another. The most quantitative approach involves the use of stable isotopes to introduce a ‘mass tag’ into the proteins from two or more different conditions and then in subsequent mass spectrometric analyses, the intensities of the differently tagged forms reflect the relative quantities in the original sample. Several such labelling methods exist and are reviewed elsewhere (Ong et al., 2003), so here we focus on the method that has been used most extensively in honey bee proteomics. It involves the reductive dimethylation of primary amines in peptides using formaldehyde isotopologues.
Assumptions: a suitable, controlled experiment should be designed to compare untreated bees/cells to equivalent samples treated with a stimulus or challenged with a pathogen. If the following steps cannot be carried out immediately, then samples can typically be stored as a cell pellet, tissue, or whole bee at -80˚C for weeks or months without protein degradation.