Multiply Charged Cation Attachment to Facilitate Mass Measurement in Negative-Mode Native Mass Spectrometry

Abstract

Native mass spectrometry (MS) is usually conducted in the positive-ion mode; however, in some cases, it is advantageous to use the negative-ion polarity. Challenges associated with native MS using ensemble measurements (i.e., the measurement of many ions at a time as opposed to the measurement of the charge and the mass-to-charge ratio of individual ions) include narrow charge state distributions with the potential for an overlap in neighboring charge states. These issues can either compromise or preclude confident charge state (and hence mass) determination. Charge state determination in challenging instances can be enabled via the attachment of multiply charged ions of opposite polarity. Multiply charged ion attachment facilitates the resolution of charge states and generates mass-to-charge (m/z) information across a broad m/z range. In this work, we demonstrated the attachment of multiply charged cations to anionic complexes generated under native MS conditions. To illustrate the flexibility available in selecting the mass and charge of the reagents, the 15+ and 20+ charge states of horse skeletal muscle apomyoglobin and the 20+ and 30+ charge states of bovine carbonic anhydrase were demonstrated to attach to model complex anions derived from either β-galactosidase or GroEL. The exclusive attachment of reagent ions is observed with no evidence for proton transfer, which is the key for the unambiguous interpretation of the post-ion/ion reaction product ion spectrum. To illustrate the application to mixtures of complex ions, the 10+ charge state of bovine ubiquitin was attached to mixtures of anions generated from the 30S and 50S particles of the Escherichia coli ribosome. Six and five major components were revealed, respectively. In the case of the 50S anion population, it was shown that the attachment of two 30+ cations of carbonic anhydrase revealed the same information as the attachment of six 10+ cations of ubiquitin. In neither case was the intact 50S particle observed. Rather, particles with different combinations of missing components were observed. This work demonstrated the utility of multiply charged cation attachment to facilitate charge state assignments in native MS ensemble measurements of heterogeneous mixtures.

Rapid and flexible online desalting using Nafion‐coated melamine sponge for mass spectrometry analysis

“…The salt cations, e.g., Na + and K + , were exchanged with H + , which would decrease the pH of the solution 66 . While many MS analyses will not be affected by the change in pH, the decrease in pH change may not be desired for applications that require the native structure of biomolecules to be maintained 71,72 and molecules that are less stable under acidic conditions. To address this issue, we first studied the pH change (ΔpH = pH (Before desalting) – pH (After desalting); Table S1, supporting information) after passing a series of 10 μM ubiquitin solutions with different salt contents including 10 mM NaCl, 0.1× PBS, and 1× PBS, through the Nafion sponge.…”

Mass spectrometry‐based methods to characterize highly heterogeneous biopharmaceuticals, vaccines, and nonbiological complex drugs at the intact‐mass level

“…Moving beyond the realm of proteins, a similar approach was used recently to obtain meaningful information on the mass distributions of oligomeric species within Tween R 80 (Betancourt et al, 2016). Lastly, a very intriguing recent development in this field was reported by McLuckey and coworkers, who used attachment of relatively small and homogeneous protein polycations to large and structurally heterogeneous polyanionic species to reduce the number of charges carried by the latter and generate the charge ladders from which the masses of the complexes (and, therefore, the polyanionic analytes) can be readily calculated (Pitts‐McCoy et al, 2022).…”

Special Issue with Research Topics on “Recent Analysis and Applications of Mass Spectra on Biochemistry”

“…Looking at soft-ionization MS methods, electrospray ionization (ESI) and matrixassisted laser/desorption ionization (MALDI) ones (ESI-and MALDI-MS) are used in many subfields of chemistry, biochemistry, and biology [81][82][83][84][85][86][87][88][89][90][91][92][93][94]. The former method generates analyte molecular-radical and protomer from solution without perturbing specific noncovalent interactions of molecular complexes, if any.…”