Investigation of the first shot phenomenon in MALDI mass spectrometry of protein complexes.
Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of noncovalent protein complexes is difficult, due to the disruptive nature of processes occurring during MALDI sample preparation and ion formation. Sometimes the observation of intact noncovalent protein complexes with MALDI is only possible if data are acquired from the first laser shot fired at a fresh sample; this is called the 'first shot phenomenon'. To study the origin of the first shot phenomenon, we used MALDI-MS and confocal laser scanning microscopy (CLSM) to examine typical MALDI sample preparations with embedded protein complexes, labeled with fluorophores. Fluorescence energy transfer techniques allowed the differentiation between intact and dissociated protein complexes with CLSM. In cases where a first shot behavior was observed by MALDI-MS, it was found to be accompanied by localization of protein complexes at the exterior of the sample crystals. Segregation of the large protein complexes to the exterior and dissociation of the complexes in the crystal interior during sample crystallization can rationalize this observation.
Wortmann
Arno
A
Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
Pimenova
Tatiana
T
Alves
Sandra
S
Zenobi
Renato
R
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D043422:Carboxypeptidases A / Q000037:antagonists & inhibitors / Q000737:chemistry; D002826:Chorismate Mutase / Q000737:chemistry; D005456:Fluorescent Dyes; D007202:Indicators and Reagents / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011506:Proteins; D019809:Streptavidin; D000426:Alcohol Dehydrogenase; D043422:Carboxypeptidases A; D002826:Chorismate Mutase