Pervasive Charge Solvation Permeates Native-like Protein Ions and Dramatically Influences Top-down Sequencing Data

Abstract

Post-translational modifications create a diverse mixture of proteoforms, leading to substantial challenges in linking proteomic information to disease. Top-down sequencing of intact proteins and multiprotein complexes offers significant advantages in proteoform analysis, but achieving complete fragmentation for such precursor ions remains challenging. Intact proteins that undergo slow-heating generally fragment via charge directed (i.e., mobile proton) or charge remote fragmentation pathways. Our efforts seek to alter this paradigm by labeling proteins with trimethyl pyrylium (TMP), which forms a stable, positively charged label at lysine residues. Fixing positive charges to the protein sequence reduces the availability of mobile protons, driving fragmentation to charge remote channels. Furthermore, we demonstrate that capping acidic side chains with carbodiimide chemistry obstructs this pathway, restoring charge-directed fragmentation and resulting in more even coverage of the protein sequence. With large amounts of fixed charge and few mobile protons, we demonstrate that it is also possible to direct fragmentation almost exclusively to lysine residues containing the charged label. Finally, our data indicate that when electrosprayed under native conditions, protein ions possess an immense capacity to accommodate excess positive charge. Molecular dynamics simulations of such ions bearing intrinsically charged labels reveal evidence of numerous charge solvation processes, including the preferential formation of helices that solvate charged labels through interactions with their macro-dipoles. Thus, these studies reveal the extent to which intact gas-phase protein ions are capable of solvating charge, and provide the most complete indication to date of the extensive physical forces opposing the goal of comprehensive top-down protein sequencing.

Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation

“…For peptides in higher charge states in which the number of protons is greater than the number of highly basic residues (Arg, Lys), protons may occupy positions along the peptide backbone, weakening amide bonds that cleave through charge-directed routes upon collisional activation. This model also explains the “proline effect” noted upon CID, as the high proton affinity of the unique proline backbone amide favors charge sequestration, consequently promoting cleavage N-terminal to proline. ,,,, For peptides in lower charge states, the ionizing protons are sequestered at side-chains with high proton affinity (i.e., Arg) and unable to migrate to facilitate backbone cleavage. In this scenario, fragmentation commonly occurs C-terminal to Asp, as acidic hydrogens on the carboxylic acid group mobilize to participate in the cleavage of the C-terminal amide bond prior to migration of more tightly sequestered protons. ,, We have already noted and discussed the probable influence of the mobile proton model on the production of b / y ions upon UVPD based on the effects of primary structure.…”

“…Re-evaluated and redefined across decades, the mobile proton model is a rigorously tested concept, producing consistent explanations for fragmentation of peptides observed by collision-induced dissociation. ,,− , Broadly, the model proposes two major mechanistic routes, classified as charge-directed and charge-remote, for peptide dissociation based on charge site localization . Governing the predominance of either dissociation route is the “mobility” of ionizing protons, in which highly mobile protons promote charge-directed fragmentation, whereas peptides with sequestered protons (nonmobile) favor charge-remote fragmentation.…”

“…This model also explains the "proline effect" noted upon CID, as the high proton affinity of the unique proline backbone amide favors charge sequestration, consequently promoting cleavage Nterminal to proline. 45,46,49,51,52 For peptides in lower charge states, the ionizing protons are sequestered at side-chains with high proton affinity (i.e., Arg) and unable to migrate to facilitate backbone cleavage. In this scenario, fragmentation commonly occurs C-terminal to Asp, as acidic hydrogens on the carboxylic acid group mobilize to participate in the cleavage of the C-terminal amide bond prior to migration of more tightly sequestered protons.…”

“…The benefits of thoroughly deciphering dissociation mechanisms have been exemplified by the landmark development of the mobile proton model to describe peptide and protein fragmentation via collision-induced dissociation . Refined across decades, this model originated from elaborate examination of the impact of residue identity, gas-phase basicity, and charge sequestration on peptide fragmentation. − Not only does the mechanism propose a robust explanation of observed fragmentation patterns of peptides, but the model has also shaped the development of bioinformatics approaches and database searches relevant for mass spectrometry-based proteomics. , Recently, evaluation of fragmentation propensities of 10,000 fragment ions produced through CID on intact proteins informed the design of a scoring mechanism tailored for native protein analysis and resulted in improved identification and characterization . Similar investigation of UVPD is envisioned to provide comparable dividends for enabling high-throughput strategies for native proteomics …”

“…45−51 Not only does the mechanism propose a robust explanation of observed fragmentation patterns of peptides, but the model has also shaped the development of bioinformatics approaches and database searches relevant for mass spectrometry-based proteomics. 51,52 Recently, evaluation of fragmentation propensities of 10,000 fragment ions produced through CID on intact proteins informed the design of a scoring mechanism tailored for native protein analysis and resulted in improved identification and characterization. 53 Similar investigation of UVPD is envisioned to provide comparable dividends for enabling high-throughput strategies for native proteomics.…”