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

Charging and dissociation of peptides and intact proteins formed by electrospray ionisation

Electrospray ionisation (ESI) mass spectrometry (MS) has emerged as a central technique for protein sequence analysis. Protein analysis by mass spectrometry typically proceeds by either the (i) top-down method, where an intact protein is ionised and fragmented inside the mass spectrometer; or (ii) the bottom-up method, where a protein is chemically digested to form peptides, which are subsequently measured by liquid chromatography (LC)-MS. However, protein and peptide ions are usually formed in low charge states and are often not ionized efficiently, which reduces the performance of MS based techniques for protein identification. Using small chemical additives (e.g. 1,2-butylene carbonate), proteins and peptides can be ‘supercharged’ to significantly improve the performance of ESI mass spectrometry for protein analysis.For top-down protein analysis, the use of 1,2-butylene carbonate and ESI can result in the formation of protein ions in sufficiently high charge states that they can protonate atmospheric gases (e.g., N2, O2, and Ar) in room-temperature ion-molecule reactions. These results suggest that protein ion charging is limited by proton transfer reactions between multiply charged protein ions and atmospheric gases. By performing ESI at reduced pressure, protein ion charge states can be increased by over 40% compared to the use of conventional chemical supercharging. The most highly charged protein ions can transfer a proton to helium, which indicates that such ions are the most acidic entities isolated to date. For bottom-up protein analysis, a T-junction was used to introduced 1,2-hexylene carbonate into the LC eluent in LC-MS measurements to significantly increase peptide ion abundances. For a whole protein HeLa digest, the average ion abundances improved by ~5.5 times from 2.2 x106 (no additive) to 1.2 x 107 (1,2-hexylene carbonate). This results in a 100% and 50% increase in the respective number of peptide and protein identifications. Using 1,2-hexylene carbonate, peptides can be identified in sub-nanogram protein loadings. These data suggesting that 1,2-hexylene carbonate will be beneficial for improving bottom-up protein analysis by LC-MS

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-channelled 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 occur in the Taylor cone prior to ESI-droplet formation, or by the fusion of droplets formed from 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 pre-mixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone, then the extent of protein charging and unfolding should be comparable or less than that obtained by pre-mixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. The presence of supercharging additives in pre-mixed 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

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

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

© 2018 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

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

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