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 highmass 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.

Native mass spectrometry of photosynthetic pigment–protein complexes

“…For MALDI, preserving non‐covalent interactions of protein complexes in the solid phase is problematic, and the singly‐charged large protein ions have m / z values usually beyond the optimum range of most commercial instruments. One prospect to overcome this is to combine cross‐linking with MALDI [19] to determine the stoichiometry, composition of protein complexes and cross‐linking yield.…”

Gold‐nanoparticle‐coated magnetic beads for concentration and ionization of analytes for laser desorption/ionization mass spectrometry

“…The ionization properties of the Au‐MAGs were exploited to ionize the dicarboxylates, which have two carboxylic acid groups in a single molecule. Such compounds with negative charges are difficult to analyze with conventional MALDI‐TOF MS . In general, the negative ion mode is less sensitive than positive ion mode producing lower intensities of ions due to the different production mechanisms for positive and negative ions.…”

“…42 Thus, in this work, the neurotransmitters and their metabolites, including ascorbic acid, norepinephrine, serotonin, and dopamine, were analyzed using the Au-MAGs without modification, as shown in The ionization properties of the Au-MAGs were exploited to ionize the dicarboxylates, which have two carboxylic acid groups in a single molecule. Such compounds with negative charges are difficult to analyze with conventional MALDI-TOF MS. [43][44][45] In general, the negative ion mode is less sensitive than positive ion mode producing lower intensities of ions due to the different production mechanisms for positive and negative ions. In this work, succinic acid (C 4 ), adipic acid (C 6 ), and suberic acid (C 8 ) were analyzed using the Au-MAGs as a solid matrix in negative mode.…”

Mass Discrimination in High-Mass MALDI-MS

“…Much more important for a good overall ion yield is the co-crystallization of the sample and the matrix molecules. It has been observed by Mädler et al that even in negative mode MALDI-MS using an acidic matrix (SA) and adding TFA, good quality spectra from basic proteins could be obtained [47]. Different proteins do not co-crystallize equally well with the matrix used.…”