RESUMEN
Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).
Asunto(s)
Biotina , Electrones , Estreptavidina , Proteínas/química , Ubiquitina/químicaRESUMEN
A quadrupoles acceptance is a measure of its ability to catch ions with certain trajectories. One way to calculate acceptance is the method of ellipses. The method arose partly from a simplification that trajectories could be calculated for an electrode axis independently of others. It has been used to calculate the acceptance and transmission of sine-driven quadrupole mass filters for over 50 years. Although the method is straightforward, it is generally described with little detail or presented as a confusing string of equations. As such, it may not be decipherable by all practitioners. For this reason, the first half of this paper presents a practical explanation of the method of ellipses and the concepts that make it work. Only equations necessary to describe the method are introduced. The tutorial also prepares the reader for the second half, which presents an alternative approach for calculating acceptance based on an array of initial trajectories. The alternative approach is used to compare the acceptance of simplified sinusoidal and digital ion guides. The method of ellipses was applied to validate results of the new approach for calculation of acceptance.