Enhancing the Quality of H/D Exchange Measurements with Mass Spectrometry Detection in Disulfide-Rich Proteins Using Electron Capture Dissociation

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a potent technique to probe higher-order structures, dynamics, and interactions of proteins. While the range of proteins amenable to interrogation by HDX MS continues to expand at an accelerating pace, there are still a few classes of proteins whose analysis with this technique remains challenging. Disulfide-rich proteins constitute one of such groups: since the reduction of thiol–thiol bonds must be carried out under suboptimal conditions (to minimize the back-exchange), it frequently results in incomplete dissociation of disulfide bridges prior to MS analysis, leading to a loss of signal, inadequate sequence coverage, and a dramatic increase in the difficulty of data analysis. In this work, the dissociation of disulfide-linked peptide dimers produced by peptic digestion of the 80 kDa glycoprotein transferrin in the course of HDX MS experiments is carried out using electron capture dissociation (ECD). ECD results in efficient cleavage of the thiol–thiol bonds in the gas phase on the fast LC time scale and allows the deuterium content of the monomeric constituents of the peptide dimers to be measured individually. The measurements appear to be unaffected by hydrogen scrambling, even when high collisional energies are utilized. This technique will benefit HDX MS measurements for any protein that contains one or more disulfides and the potential gain in sequence coverage and spatial resolution would increase with disulfide bond number

A New Approach to Measuring Protein Backbone Protection with High Spatial Resolution Using H/D Exchange and Electron Capture Dissociation

Inadequate spatial resolution remains one of the most serious limitations of hydrogen/deuterium exchange-mass spectrometry (HDX-MS), especially when applied to larger proteins (over 30 kDa). Supplementing proteolytic fragmentation of the protein in solution with ion dissociation in the gas phase has been used successfully by several groups to obtain near-residue level resolution. However, the restrictions imposed by the LC–MS/MS mode of operation on the data acquisition time frame makes it difficult in many cases to obtain a signal-to-noise ratio adequate for reliable assignment of the backbone amide protection levels at individual residues. This restriction is lifted in the present work by eliminating the LC separation step from the workflow and taking advantage of the high resolving power and dynamic range of a Fourier transform ion cyclotron resonance-mass spectrometer (FTICR-MS). A residue-level resolution is demonstrated for a peptic fragment of a 37 kDa recombinant protein (N-lobe of human serum transferrin), using electron-capture dissociation as an ion fragmentation tool. The absence of hydrogen scrambling in the gas phase prior to ion dissociation is verified using redundant HDX-MS data generated by FTICR-MS. The backbone protection pattern generated by direct HDX-MS/MS is in excellent agreement with the known crystal structure of the protein but also provides information on conformational dynamics, which is not available from the static X-ray structure

Mass Spectrometry-Guided Optimization and Characterization of a Biologically Active Transferrin–Lysozyme Model Drug Conjugate

Transferrin is a promising drug carrier that has the potential to deliver metals, small organic molecules and therapeutic proteins to cancer cells and/or across physiological barriers (such as the blood–brain barrier). Despite this promise, very few transferrin-based therapeutics have been developed and reached clinical trials. This modest success record can be explained by the complexity and heterogeneity of protein conjugation products, which also pose great challenges to their analytical characterization. In this work, we use lysozyme conjugated to transferrin as a model therapeutic that targets the central nervous system (where its bacteriostatic properties may be exploited to control infection) and develop analytical protocols based on electrospray ionization mass spectrometry to characterize its structure and interactions with therapeutic targets and physiological partners critical for its successful delivery. Mass spectrometry has already become an indispensable tool facilitating all stages of the protein drug development process, and this work demonstrates the enormous potential of this technique in facilitating the development of a range of therapeutically effective protein–drug conjugates

A New Approach to Measuring Protein Backbone Protection with High Spatial Resolution Using H/D Exchange and Electron Capture Dissociation

Inadequate spatial resolution remains one of the most serious limitations of hydrogen/deuterium exchange-mass spectrometry (HDX-MS), especially when applied to larger proteins (over 30 kDa). Supplementing proteolytic fragmentation of the protein in solution with ion dissociation in the gas phase has been used successfully by several groups to obtain near-residue level resolution. However, the restrictions imposed by the LC–MS/MS mode of operation on the data acquisition time frame makes it difficult in many cases to obtain a signal-to-noise ratio adequate for reliable assignment of the backbone amide protection levels at individual residues. This restriction is lifted in the present work by eliminating the LC separation step from the workflow and taking advantage of the high resolving power and dynamic range of a Fourier transform ion cyclotron resonance-mass spectrometer (FTICR-MS). A residue-level resolution is demonstrated for a peptic fragment of a 37 kDa recombinant protein (N-lobe of human serum transferrin), using electron-capture dissociation as an ion fragmentation tool. The absence of hydrogen scrambling in the gas phase prior to ion dissociation is verified using redundant HDX-MS data generated by FTICR-MS. The backbone protection pattern generated by direct HDX-MS/MS is in excellent agreement with the known crystal structure of the protein but also provides information on conformational dynamics, which is not available from the static X-ray structure

LC/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC/MS and cross-path reactive chromatography (XP-RC) MS

Interfacing liquid chromatography (LC) with electrospray ionization (ESI) to enable on-line MS detection hadbeen initially implemented using reversed phase LC, which in the past three decades remained the default type ofchromatography used for LC/MS and LC/MS/MS studies of protein structure. In contrast, the advantages of othertypes of LC as front-ends for ESI MS, particularly those that allow biopolymer higher order structure to bepreserved throughout the separation process, enjoyed relatively little appreciation until recently. However, thepast few years witnessed a dramatic surge of interest in the so-called “native” (with “non-denaturing” beingperhaps a more appropriate adjective) LC/MS and LC/MS/MS analyses within the bioanalytical and biophysicalcommunities. This review focuses on recent advances in this field, with an emphasis on size exclusion and ionexchange chromatography as front-end platforms for protein characterization by LC/MS. Also discussed are thebenefits provided by the integration of chemical reactions in the native LC/MS analyses, including both ionchemistry in the gas phase (e.g., limited charge reduction for characterization of highly heterogeneous biopolymers)and solution-phase reactions (using the recently introduced technique cross-path reactive chromatography)

Human Serum Transferrin: Is There a Link among Autism, High Oxalate Levels, and Iron Deficiency Anemia?

It has been previously suggested that large amounts of oxalate in plasma could play a role in autism by binding to the bilobal iron transport protein transferrin (hTF), thereby interfering with iron metabolism by inhibiting the delivery of iron to cells. By examining the effect of the substitution of oxalate for the physiologically utilized synergistic carbonate anion in each lobe of hTF, we sought to provide a molecular basis for or against such a role. Our work clearly shows both qualitatively (6 M urea gels) and quantitatively (kinetic analysis by stopped-flow spectrofluorimetry) that the presence of oxalate in place of carbonate in each binding site of hTF does indeed greatly interfere with the removal of iron from each lobe (in the absence and presence of the specific hTF receptor). However, we also clearly demonstrate that once the iron is bound within each lobe of hTF, neither anion can displace the other. Additionally, as verified by urea gels and electrospray mass spectrometry, formation of completely homogeneous hTF–anion complexes requires that all iron must first be removed and hTF then reloaded with iron in the presence of either carbonate or oxalate. Significantly, experiments described here show that carbonate is the preferred binding partner; i.e., even if an equal amount of each anion is available during the iron loading process, the hTF–carbonate complex is formed

Structural and functional consequences of the substitution of glycine 65 with arginine in the N-lobe of human transferrin

The G65R mutation in the N-lobe of human transferrin was created to mimic a naturally occurring variant (G394R) found in the homologous C-lobe. Because Gly65 is hydrogen-bonded to the iron-binding ligand Asp63, it comprises part of the second shell hydrogen bond network surrounding the iron within the metal binding cleft of the protein. Substitution with an arginine residue at this position disrupts the network, resulting in much more facile removal of iron from the G65R mutant. As shown by UV-vis and EPR spectroscopy, and by kinetic assays measuring the release of iron, the G65R mutant can exist in three forms. Two of the forms (yellow and pink in color) are inter-convertible. The yellow form predominates in 1 M bicarbonate; the pink form is generated from the yellow form upon exchange into 1 M HEPES buffer, pH 7.4. The third form (also pink in color) is produced by the addition of Fe(3+)-(nitrilotriacetate)(2) to apo-G65R. Hydrogen/deuterium exchange experiments are consistent with all forms of the G65R mutant assuming a more open conformation. Additionally, mass spectroscopic analysis reveals the presence of nitrilotriacetate in the third form. The inability to obtain crystals of the G65R mutant, led to development of a novel crystallization strategy in which the double mutation G65R/K206E stabilizes a single closed pink conformer and captures Arg65 in a single position. Collectively, these studies highlight the importance of the hydrogen bond network in the cleft, as well as the inherent flexibility of the N-lobe which although able to adapt to accommodate the large arginine substitution exists in multiple conformations