Localization of Noncovalent Complexes in MALDI-Preparations by CLSM

The unambiguous detection of noncovalent complexes (NCCs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still a far cry from being routine. For protein NCCs such as their quaternary structure it has been reported that signals of the intact complex are only obtained for the first or at most the first few laser exposures of a given sample area. This observation was called the first-shot phenomenon. In the present study, this first-shot phenomenon has been investigated for the hexameric protein complex allophycocyanine (APC) by two independent methods, MALDI-MS with a (nearly) pH-neutral matrix 6-aza-2-thiothymine (6-ATT) and by imaging the fluorescence of the complex in APC-6-ATT preparations by confocal laser scan microscopy (CLSM). The intact APC heterohexamer loses its visible fluorescence upon dissociation into its subunits. Both methods consistently show that intact APC complexes are precipitated at the matrix crystal surface, but dissociate upon incorporation into the matrix crystals.