184.108.40.206. Mass spectrometry analysis
the combined sample with at least a two-hour gradient into a liquid
chromatography-tandem mass spectrometry system (LC-MS/MS).
Most labs will access such a system through a core facility rather than operating the system themselves. Interested readers are referred elsewhere for details on setting up such a system (Forner et al., 2007).
- Search the LC-MS/MS data against a protein database using MaxQuant (Cox and Mann, 2008), Proteome Discoverer (from ThermoFisher) or SpectrumMill (from Agilent).
Background on the principles underlying such database searching has been covered elsewhere (Forner et al., 2007). The database should contain all protein sequences from all species that might be in the sample. For example, if the experiment has involved infecting bees with P. larvae, the database should contain all A. mellifera and all P. larvae sequences available from their respective genome project pages in GenBank. The search engines mentioned above should also report the isotope ratios representing the relative amounts of each protein detected among the different samples.