title: Compelling advantages of negative ion mode detection in high-mass MALDI-MS for homomeric protein complexes.
abstract: Chemical cross-linking in combination with high-mass MALDI mass spectrometry allows for the rapid identification of interactions and determination of the complex stoichiometry of noncovalent protein-protein interactions. As the molecular weight of these complexes increases, the fraction of multiply charged species typically increases. In the case of homomeric complexes, signals from multiply charged multimers overlap with singly charged subunits. Remarkably, spectra recorded in negative ion mode show lower abundances of multiply charged species, lower background, higher reproducibility, and, thus, overall cleaner spectra compared with positive ion mode spectra. In this work, a dedicated high-mass detector was applied for measuring high-mass proteins (up to 200 kDa) by negative ion mode MALDI-MS. The influences of sample preparation and instrumental parameters were carefully investigated. Relative signal integrals of multiply charged anions were relatively independent of any of the examined parameters and could thus be approximated easily for the spectra of cross-linked complexes. For example, the fraction of doubly charged anions signals overlapping with the signals of singly charged subunits could be more precisely estimated than in positive ion mode. Sinapinic acid was found to be an excellent matrix for the analysis of proteins and cross-linked protein complexes in both ion modes. Our results suggest that negative ion mode data of chemically cross-linked protein complexes are complementary to positive ion mode data and can in some cases represent the solution phase situation better than positive ion mode.
authors: Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
authors: Stefanie
authors: S
authors: Mädler
authors: Konstantin
authors: K
authors: Barylyuk
authors: Elisabetta
authors: E
authors: Boeri Erba
authors: Robert J
authors: RJ
authors: Nieckarz
authors: Renato
authors: R
authors: Zenobi
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry*; D002417:Cattle; D002645:Chickens; D006736:Horses; D006863:Hydrogen-Ion Concentration; D010710:Phosphates; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000838:Anions; D010710:Phosphates; D021122:Protein Subunits; D011506:Proteins; C018279:sodium phosphate