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, β-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

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

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

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 complimentary 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.</p

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

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 (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis. 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 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 pKa 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 pKa 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. Keywords: Electrospray ionisation, Mass spectrometry, Protein ions, Supercharging, Ion evaporation model, Charge residue model, Chain ejection model, 1,2-butylene carbonate, 1,2-hexylene carbonate, 1,2-propylene carbonat

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

Internal Fragments Generated by Electron Ionization Dissociation Enhances 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 (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown 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 do not contain the C-terminus or 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. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments

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 (Ubq_1–11) 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, β-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