Identification of components of protein complexes by mass spectrometry has become an important and powerful approach to understand cell biology. Tandem mass spectrometry can provide unbiased and comprehensive identification of proteins by generating high accuracy mass information for peptide ions in MS1 spectra and further amino acid sequence information is reported as fragment ion information in MS2 spectra. The combination of this data can be used to confidently identify peptides and thus protein matches from sequence databases. The protocol described here is applicable to any protein complex that can be isolated to sufficient purity and quantity, its components separated by gel electrophoresis and sequence databases for the source organism are available. In the referenced Nature Neuroscience paper we used this approach to identify 220 proteins associated with the glutamate receptor NR2 subunit in D. melanogaster. Complexes were isolated using a hexapeptide corresponding to the C-terminus of NR2 that contains a PDZ protein interaction motif. Comparison of this NR2-associated protein complex in D. melanogaster with that characterised by the same purification in M. musculus revealed species-specific adaptation with greater signalling complexity in mouse.