The extent of multiple charging protein ions in electrospray ionization (ESI) mass spectra depends on the solvent-exposed surface area, but it may also be influenced by a variety other extrinsic and intrinsic factors. Gas-phase ion chemistry (charge-transfer charge-partitioning reactions) appears to major factor influencing protonation as detected ESI MS. In this work, we demonstrate that under carefully controlled conditions, which limit occurrence charge-transfer reactions gas phase,...

Monitoring the changes in charge-state distributions of protein ions electrospray ionization (ESI) mass spectra has become one commonly accepted tools to detect large-scale conformational proteins solution. However, these experiments produce only qualitative, low-resolution information. Our goal is develop a procedure that would quantitative data on isomers coexisting solution at equilibrium. To end, we have examined evolution positive ion ESI two model proteins, α-helical myoglobin (Mb) and...

Multiple charging is an intrinsic feature of electrospray ionization (ESI) macromolecules. While multiple factors influence the appearance protein ion charge state distributions in ESI mass spectra, physical dimensions molecules solution are major determinants extent charging. This article reviews information that can be obtained by analyzing ionic MS, as well potential pitfalls and limitations this powerful technique. We also discuss future areas growth with particular emphasis on...

Electron-transfer dissociation (ETD) is evaluated as a technique to provide local information on higher order structure and dynamics of whole protein molecule. Isotopic labeling highly flexible segments model 18 kDa carried out in solution under mildly denaturing conditions by means hydrogen/deuterium exchange (HDX), followed transfer intact ions the gas phase electrospray ionization, mass-selection precursor ion for subsequent reactions with fluoranthene radical anions. The ETD process...

Hydrogen/deuterium exchange (HDX) combined with mass spectrometry (MS) detection has matured in recent years to become a powerful tool structural biology and biophysics. Several limitations of this technique can will be addressed by tapping into the ever expanding arsenal methods manipulate ions gas phase offered spectrometry.

Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due the structural heterogeneity inherent many protein-based therapeutics (e.g., extensive glycosylation or "designer" modifications such as chemical conjugation) introduced postproduction result stress oxidation deamidation). A combination ion-exchange chromatography...

Abstract Conformational heterogeneity of α‐synuclein was studied with electrospray ionization mass spectrometry by analyzing protein ion charge state distributions, where the extent multiple charging reflects compactness conformations in solution. Although lacks a single well‐defined structure under physiological conditions, it found to sample four distinct conformational states, ranging from highly structured one random coil. The compact is present across entire range conditions tested (pH...

Self-association of proteins is important in a variety processes ranging from acquisition native quaternary structure (where the association tightly controlled and proceeds highly ordered fashion) to aggregation amyloidosis. The latter frequently accompanied (or indeed triggered) by loss structure, but clear understanding complex relationship between conformational changes protein self-association/aggregation remains elusive due great difficulty characterizing these heterogeneous species. In...

Bovine β-lactoglobulin (β-Lg) is one of major allergens in cow's milk. Previous study showed that ultrasound treatment induced the conformational changes β-Lg and promoted glycation aqueous solutions, which is, however, less efficient compared with dry-state. In this work, effect pretreatment combined dry-state on IgG IgE binding was studied. Dry-state mannose after at 0-600 W significantly reduced β-Lg, lowest values observed 400 W. The decrease attributed to increase extent secondary...

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over 6,000,000 cases nearly 400,000 deaths reported worldwide by end May 2020. A rush to find cure prompted re-evaluation range existing therapeutics vis-à-vis their potential role treating COVID-19, placing premium on analytical tools capable supporting such efforts. Native mass spectrometry (MS) has long been tool choice mechanistic studies drug/therapeutic target interactions,...

Protein amide hydrogen exchange (HDX) is a convoluted process, whose kinetics determined by both dynamics of the protein and intrinsic rate labile atoms fully exposed to solvent. Both processes are influenced variety extrinsic factors. A mathematical formalism initially developed rationalize individual now often used interpret global (e.g., as measured in HDX MS experiments). One particularly important advantage direct visualization various states observing distinct ion populations with...

Mass spectrometry plays a very visible role in biopharmaceutical industry, although its use development, characterization, and quality control of protein drugs is mostly limited to the analysis covalent structure (amino acid sequence post-translational modifications). Despite centrality conformation biological activity, stability, safety products, expanding arsenal mass spectrometry-based methods that are currently available probe higher order conformational dynamics biopolymers did not,...

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety factors that not solely defined their covalent chemical structure. Since conformation (or higher order structure) is major modulator its biological activity, ability to detect changes both structure and dynamics protein, induced an array extrinsic factors, central importance producing, purifying, formulating commercial biopharmaceutical with consistent therapeutic...

Dynamics of bovine hemoglobin assembly was investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. Intensities ionic signals corresponding to various protein species (tetramers, dimers, heme-deficient as well apo- holo-monomers) were used estimate relative fractions these a function pH. The fraction folded for each observed estimated based on charge-state distributions the spectra. cumulative...

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide wealth of information on protein dynamics, because they contain contributions from all conformers present solution. Such ionic often overlap, limiting the amount useful that can be extracted spectra. This difficulty is overcome work by using chemometric approach, which allows spectral deconvolution carried out and individual extracted. Experiments are acquiring series over range...

The extent of internal hydrogen exchange (scrambling) within multiply charged solvent-free protein ions was investigated using a small model protein. site-specific backbone amide protection data were obtained ion fragmentation in the gas phase and compared with available NMR data. Only minimal scrambling detected when relatively high-energy collisional activation used to fragment intact ions, while low-energy resulted more significant but not random exchange. Increased conformational...