Stefanie Mädler1, Konstantin Barylyuk1, Elisabetta Boeri Erba1, Robert J. Nieckarz1, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
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.
CovalX Technology Used (Click each option to learn more)
Using columns with a molecular weight cut-off of 50 kDa and 10 mM phosphate buffer (NaH2PO4-Na2HPO4, pH 8), protein samples in ammonium buffers (citrate synthase, pyruvate kinase, aldolase) were ultrafiltered multiple times. Protein concentration, type of crosslinker and molar excess were all optimized individually for use with citrate synthase, aldolase, GST and GADPH. 10 μL of protein solutions at 5 to 40 μM were mixed with a 40- to 100-fold molar excess of DSS, BS3, or SBAT dissolved in dimethylformamide (DSS, SBAT) or water (BS3) in a 10/1 (v/v) ratio and incubated for 0.5 to 2 hours at room temperature. Samples were mixed with a matrix (sinapic acid dissolved at 10 mg/mL in water/acetonitrile/TFA (49.95/49.95/0.1, v/v/v) in a 1/1 vikyne ratio. 1 μL of the final mixture was spotted on a stainless steel plate and dried under ambient conditions before being analyzed by a mass spectrometer that had been modified with a CovalX HM2 detection system. The CovalX Complex Tracker software was used to interpret the data.