Digital Ion Trap Isolation and Mass Analysis of Macromolecular Analytes with Multiply Charged Ion Attachment.

Multiply charged ions produced by electrospray ionization (ESI) of heterogeneous mixtures of macromolecular analytes under native conditions are typically confined to relatively narrow ranges of mass-to-charge (m/z) ratio, often with extensive overlap. This scenario makes charge and mass assignments extremely challenging, particularly when individual charge states are unresolved. An ion/ion reaction strategy involving multiply charged ion attachment (MIA) to the mixture components in a narrow range of m/z can facilitate charge and mass assignment. In MIA operation, multiply charged reagent ions are attached to the analyte ions of opposite polarity to provide large m/z displacements resulting from both large changes in mass and charge. However, charge reduction of the high m/z ions initially generated under native ESI conditions requires the ability to isolate high m/z ions and to analyze even higher m/z product ions. Digital ion trap (DIT) operation offers means for both high m/z ion isolation and high m/z mass analysis, in addition to providing conditions for the reaction of oppositely charged ions. The feasibility of conducting MIA experiments in a DIT that takes advantage of high m/z ion operation is demonstrated here using a tandem 2D-3D DIT instrument. Proof-of-concept MIA experiments with cations derived from ß-galactosidase using the 20- charge state of human serum immunoglobulin G (IgG, ∼149 kDa) as the reagent anion are described. MIA experiments involving mixtures of ions derived from the E. coli. ribosome are also described. For example, three components in a mixture of 70S particles (>2.2 MDa) were resolved and assigned with masses and charges following an MIA experiment involving the 20- charge state of human serum IgG.

Submicron Emitters Enable Reliable Quantification of Weak Protein-Glycan Interactions by ESI-MS.

Interactions between carbohydrates (glycans) and glycan-binding proteins (GBPs) regulate a wide variety of important biological processes. However, the affinities of most monovalent glycan-GBP complexes are typically weak (dissociation constant (Kd) > µM) and difficult to reliably measure with conventional assays; consequently, the glycan specificities of most GBPs are not well established. Here, we demonstrate how electrospray ionization mass spectrometry (ESI-MS), implemented with nanoflow ESI emitters with inner diameters of ∼50 nm, allows for the facile quantification of low-affinity glycan-GBP interactions. The small size of the droplets produced from these submicron emitters effectively eliminates the formation of nonspecific glycan-GBP binding (false positives) during the ESI process up to ∼mM glycan concentrations. Thus, interactions with affinities as low as ∼5 mM can be measured directly from the mass spectrum. The general suppression of nonspecific adducts (including nonvolatile buffers and salts) achieved with these tips enables ESI-MS glycan affinity measurements to be performed on C-type lectins, a class of GBPs that bind glycans in a calcium-dependent manner and are important regulators of immune response. At physiologically relevant calcium ion concentrations (2-3 mM), the extent of Ca2+ nonspecific adduct formation observed using the submicron emitters is dramatically suppressed, allowing glycan affinities, and the influence of Ca2+ thereon, to be measured. Finally, we show how the use of submicron emitters and suppression of nonspecific binding enable the quantification of labile (prone to in-source dissociation) glycan-GBP interactions.