On the Mechanism of Theta Capillary Nanoelectrospray Ionization for the Formation of Highly Charged Protein Ions Directly from Native Solutions.

Theta capillary nanoelectrospray ionization (θ-nanoESI) can be used to "supercharge" protein ions directly from solution for detection by mass spectrometry (MS). In native top-down MS, the extent of protein charging is low. Given that ions with more charge fragment more readily, increasing charge can enhance the extent of sequence information obtained by top-down MS. For θ-nanoESI, dual-channeled nanoESI emitters are used to mix two solutions in low to sub-µs prior to MS. The mechanism for θ-nanoESI mixing has been reported to primarily occur: (i) in a single shared Taylor cone and in the droplets formed from the Taylor cone or (ii) by the fusion of droplets formed from two separate Taylor cones. Using θ-nanoESI-ion mobility MS, native protein solutions were rapidly mixed with denaturing supercharging solutions to form protein ions in significantly higher charge states and with more elongated structures than those formed by premixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone and in the droplets resulting from the single Taylor cone, then the extent of protein charging and unfolding should be comparable to or less than that obtained by premixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. These data also suggest that highly charged protein ions can be formed by the near-complete mixing of each solution. The presence of supercharging additives in premixed solutions can suppress volatile electrolyte evaporation, limiting the extent of protein charging compared to when the additive is delivered via one channel of a θ-nanoESI emitter. In θ-nanoESI, the formation of two Taylor cones can presumably result in substantial electrolyte evaporation from the ESI droplets containing native-like proteins prior to droplet fusion, thereby enhancing ion charging.

Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins.

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20-60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody-drug conjugates.

ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry.

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789.

Extended Protein Ions Are Formed by the Chain Ejection Model in Chemical Supercharging Electrospray Ionization.

Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq1-11) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, ß-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model.

Highly Charged Protein Ions: The Strongest Organic Acids to Date.

The basicity of highly protonated cytochrome c (cyt c) and myoglobin (myo) ions were investigated using tandem mass spectrometry, ion-molecule reactions (IMRs), and theoretical calculations as a function of charge state. Surprisingly, highly charged protein ions (HCPI) can readily protonate non-polar molecules and inert gases, including Ar, O2 , and N2 in thermal IMRs. The most HCPIs that can be observed are over 130 kJ mol-1 less basic than the least basic neutral organic molecules known (tetrafluoromethane and methane). Based on theoretical calculations, it is predicted that protonated cyt c and myo ions should spontaneously lose a proton to vacuum for charge states in which every third residue is protonated. In this study, HCPIs are formed where every fourth residue on average is protonated. These results indicate that protein ions in higher charge states can be formed using a low-pressure ion source to reduce proton-transfer reactions between protein ions and gases from the atmosphere.

Extremely supercharged proteins in mass spectrometry: profiling the pH of electrospray generated droplets, narrowing charge state distributions, and increasing ion fragmentation.

The effects of 12 acids, 4 solvents, and 8 low-volatility additives that increase analyte charging (i.e., superchargers) on the charge state distributions (CSDs) of protein ions in ESI-MS were investigated. We discovered that (i) relatively low concentrations [5% (v/v)] of 1,2-butylene carbonate (and 4-vinyl-1,3-dioxolan-2-one) can be added to ESI solutions to form higher charge states of cytochrome c and myoglobin ions than by using more traditional additives (e.g., propylene carbonate, sulfolane, or m-nitrobenzyl alcohol) under these conditions and (ii) the width of CSDs narrow as the effectiveness of superchargers increase, which concentrates protein ions into fewer detection channels. The use of strong acids (pKa values < 0) results in essentially no protein supercharging, higher adduction of acid molecules, and wider CSDs for many superchargers and proteins, whereas the use of weak acids (pKa > 0) results in significantly higher protein ion charging, less acid adduction, and narrower CSDs, indicating that protein ion supercharging in ESI can be significantly limited by the binding of conjugate base anions of acids that neutralize charge sites and broaden CSDs. The extent of protein charging as a function of acid identity (HA) does not strongly correlate with gas-phase proton transfer data (i.e., gas-phase basicity and proton affinity values for HA and A(-)), solution-phase protein secondary structures (as determined by circular dichroism spectroscopy), and/or acid molecule volatility data. For protein-denaturing solutions, these data were used to infer that the "effective" pH of ESI generated droplets near the moment of ion formation can be ∼0, which is ca. 1 to 3 pH units lower than the pH of the solutions prior to ESI. Electron capture dissociation (ECD) of [ubiquitin, 17H](17+) resulted in the identification of 223 cleavages, 74 of 75 inter-residue sites, and 92% ECD fragmentation efficiency, which correspond to highest of these values that have been obtained by ECD of a single isolated charge state of ubiquitin.

Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition.

Parkinson's disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) top-down (TD) mass spectrometry of α-synuclein/CLR01 complexes pinpoints the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggest that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.

Towards understanding the formation of internal fragments generated by collisionally activated dissociation for top-down mass spectrometry.

Top-down mass spectrometry (TD-MS) generates fragment ions that returns information on the polypeptide amino acid sequence. In addition to terminal fragments, internal fragments that result from multiple cleavage events can also be formed. Traditionally, internal fragments are largely ignored due to a lack of available software to reliably assign them, mainly caused by a poor understanding of their formation mechanism. To accurately assign internal fragments, their formation process needs to be better understood. Here, we applied a statistical method to compare fragmentation patterns of internal and terminal fragments of peptides and proteins generated by collisionally activated dissociation (CAD). Internal fragments share similar fragmentation propensities with terminal fragments (e.g., enhanced cleavages N-terminal to proline and C-terminal to acidic residues), suggesting that their formation follows conventional CAD pathways. Internal fragments should be generated by subsequent cleavages of terminal fragments and their formation can be explained by the well-known mobile proton model. In addition, internal fragments can be coupled with terminal fragments to form complementary product ions that span the entire protein sequence. These enhance our understanding of internal fragment formation and can help improve sequencing algorithms to accurately assign internal fragments, which will ultimately lead to more efficient and comprehensive TD-MS analysis of proteins and proteoforms.

Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan.

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

Naked Mole-Rat, a Rodent with an Apolipoprotein A-I Dimer.

A variety of rodents have been used as experimental animals in metabolic studies of plasma lipids and lipoproteins. These studies have included understanding the functional role of apolipoprotein A-I, the major protein on the surface of HDL. Reviewing the genomic database for entries for rodent apoA-I genes, it was discovered that the naked mole-rat (Heterocephalus glaber) gene encoded a protein with a cysteine at residue 28. Previously, two cases have been reported in which human heterozygotes had apoA-I with cysteine at residues 173 (apoA-I Milano) or at 151 (apoA-I Paris). Interestingly, both groups, in spite of having low levels of HDL and moderately elevated plasma triacylglycerols, had no evidence of cardiovascular disease. Moreover, the presence of the cysteine enabled the apoA-I to form both homodimers and heterodimers. Prior to this report, no other mammalian apoA-I has been found with a cysteine in its sequence. In addition, the encoded naked mole-rat protein had different amino acids at sites that were conserved in all other mammals. These differences resulted in naked mole-rat apoA-I having an unexpected neutral pI value, whereas other mammalian apoA-I have negative pI values. To verify these sequence differences and to determine if the N-terminal location of C28 precluded dimer formation, we conducted mass spectrometry analyses of apoA-I and other proteins associated with HDL. Consistent with the genomic data, our analyses confirmed the presence of C28 and the formation of a homodimer. Analysis of plasma lipids surprisingly revealed a profile similar to the human heterozygotes.

Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence Coverage.

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N- or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photon-based fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15-20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry.

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ∼20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ∼50 to ∼99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.

On the mechanism of protein supercharging in electrospray ionisation mass spectrometry: Effects on charging of additives with short- and long-chain alkyl constituents with carbonate and sulphite terminal groups.

Small organic molecules are used as solution additives in electrospray ionisation mass spectrometry (ESI-MS) to increase the charge states of protein ions and improve the performance of intact protein analysis by tandem mass spectrometry. The properties of the additives that are responsible for their charge-enhancing effects (e.g. dipole moment, gas-phase basicity, Brønsted basicity, and surface tension) have been debated in the literature. We report a series of solution additives for ESI-MS based on cyclic alkyl carbonates and sulphites that have alkyl chains that are from two to ten methylene units long. The extent of charging of [Val [5]]-angiotensin II, cytochrome c, carbonic anhydrase II, and bovine serum albumin in ESI-MS using the additives was measured. For both the alkyl carbonate and sulphite additives with up to four methylene units, ion charging increased as the side chain lengths of the additives increased. At a critical alkyl chain length of four methylene units, protein ion charge states decreased as the chain length increased. The dipole moments, gas-phase basicity values, and Brønsted basicities (i.e. the pK a of the conjugate acids) of the additives were obtained using electronic structure calculations, and the surface tensions were measured by pendant drop tensiometry. Because the dipole moments, gas-phase basicities, and pK a values of the additives did not depend significantly on the alkyl chain lengths of the additives and the extent of charging depended strongly on the chain lengths, these data indicate that these three additive properties do not correlate with protein charging under these conditions. For the additives with alkyl chains at or above the critical length, the surface tension of the additives decreased as the length of the side chain decreased, which correlated well with the decrease in protein charging. These data are consistent with protein charging being limited by droplet surface tension below a threshold surface tension for these additives. For additives with relatively high surface tensions, protein ion charging increased as the amphiphilicity of the additives increased (and surface tension decreased) which is consistent with protein charging being limited by the emission of charge carriers from highly charged ESI generated droplets.

Supercharging protein ions in native mass spectrometry using theta capillary nanoelectrospray ionization mass spectrometry and cyclic alkylcarbonates.

Theta nanoelectrospray ionization of protein ions formed from aqueous buffer solutions that are mixed with denaturing solutions containing cyclic alkylcarbonates (e.g., vinyl ethylene carbonate; VEC) results in a significant increase in the extent of ion charging compared to native mass spectrometry. For six proteins, the extent of ion charging can be significantly higher than that obtained using denaturing solutions and alternative native "supercharging" methods. In theta nanoelectrospray supercharging, the extent of charging scales with protein mass in agreement with an analytical scaling relationship for ions with elongated structures. Theta nanoelectrospray supercharging of non-covalent complexes from native solutions results in essentially the complete loss of protein-ligand and protein-protein interactions. Based on circular dichroism spectroscopy, VEC can effectively denature proteins in buffered solutions. These data provide evidence that enrichment of VEC in theta nanoelectrospray ionization generated droplets can denature proteins on the timescale of droplet desolvation and ion formation. This approach can be used to form highly charged protein ions from native solutions containing biological buffers, including some that are considered incompatible with native MS. Forming some protein ions in the highest reported charge states directly from native solutions is no longer a challenge in obtaining primary structural information using tandem mass spectrometry.