Decoding Protein Gas‐Phase Stability with Alanine Scanning and Collision‐Induced Unfolding Ion Mobility Mass Spectrometry

Bellamy‐Carter, Jeddidiah; O'Grady, Louisa; Passmore, Munro; Jenner, Matthew; Oldham, Neil J.

doi:10.1002/anse.202000019

Cited by 6 publications

(11 citation statements)

References 52 publications

(105 reference statements)

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“…The 5+ ion was then generated by leaving E18 deprotonated (Figure S12b), as – of the overall neutral acidic residues in the 6+ structure – this position showed the highest probability of carrying a charge by being deprotonated. Recently we have developed a Python‐based charge placement algorithm entitled ‘ChargePlacer’ which provides a general tool for predicting charge sites on protein ions [36] . Using the charge sites listed above, MD simulations were run either at 298 K to produce low energy gas‐phase structures, or 750 K in order to induce unfolding.…”

Section: Resultsmentioning

confidence: 99%

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility‐Mass Spectrometry Provides Insights into Protein Gas‐Phase Structure

Al-jabiry

Palmer

Langridge

et al. 2021

Chemistry A European J

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Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas‐phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility‐mass spectrometry (IM‐MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas‐phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N‐terminus and E22 in a ubiquitin binding protein UBA2.

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Section: Resultsmentioning

confidence: 99%

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility‐Mass Spectrometry Provides Insights into Protein Gas‐Phase Structure

Al-jabiry

Palmer

Langridge

et al. 2021

Chemistry A European J

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“…Our simulations suggest this stability stems from the scarcity of carboxylate side chains in GFP Bas , making the protons distribute over the high number of basic residues for a favorable Coulombic energy. It seems likely that the larger number of basic residues in this variant also allows for a more optimal distribution of charges that minimizes repulsion and creates more opportunities for salt bridges that stabilize compact states in the gas phase. , The importance of charge locations is emphasized by the different conformational preferences of TTHA variants in the gas phase. Furthermore, the combination of basic and acidic residues raises the possibility of salt bridge formation on the protein surface, which can also alter gas phase stability …”

Section: Discussionmentioning

confidence: 99%

“… 15 By combining alanine scanning with CIU, it is possible to quantify the contributions of individual residues on the conformational stabilities of protein ions. 16 IM-MS is furthermore employed in the pharmaceutical industry, 17 for example to characterize drug conjugates. 18 …”

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confidence: 99%

“…IM-MS has been successfully applied to characterize the lipid preferences of membrane proteins, , define structural heterogeneity associated with glycosylation, follow structural transitions in disordered proteins, and determine the impact of disease-associated mutations on protein stability . By combining alanine scanning with CIU, it is possible to quantify the contributions of individual residues on the conformational stabilities of protein ions . IM-MS is furthermore employed in the pharmaceutical industry, for example to characterize drug conjugates …”

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confidence: 99%

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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Abramsson

Sahin

Hopper

et al. 2021

JACS Au

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In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

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“…The thermal conductivity of the buffer gas determines its ability to transfer energy , to the analyte upon collision, which can affect not only T but the three-dimensional structure. Extensive heat transfer can cause unfolding or fragmentation of the analyte. − As an example, He has a larger thermal conductivity than N 2 and thus produces more heat upon interactions with an analyte, affecting T . The characteristics of the different drift gases are often overlooked as well.…”

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Propagating Error through Traveling-Wave Ion Mobility Calibration

Edwards

Tran

Gallagher

2021

J. Am. Soc. Mass Spectrom.

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Native mass spectrometry (MS) is used to elucidate the stoichiometry of protein complexes and quantify binding interactions by maintaining native-like, noncovalent interactions in the gas phase. However, ionization forces proteins into specific conformations, losing the solution-phase dynamics associated with solvated protein structures. Comparison of gas-phase structures to those in solution, or to other gas-phase ion populations, has many biological implications. For one, analyzing the variety of conformations that are maintained in the gas-phase can provide insight into a protein's solution-phase energy landscape. The gasphase conformations of proteins and complexes can be investigated using ion mobility (IM) spectrometry. Specifically, drift tube (DT)-IM utilizes uniform electric fields to propel a population of gas-phase ions through a region containing a neutral gas. By measuring the mobility (K) of gas-phase ions, users are able to calculate an average momentum transfer cross section ( DT CCS), which provides structural information on the ion. Conversely, in traveling-wave ion mobility spectrometry (TWIMS), TW CCS values cannot be derived directly from an ion's mobility but must be determined following calibration. Though the required calibration adds uncertainty, it is common to report only an average and standard deviation of the calculated TW CCS, accounting for uncertainty associated with replicate measurements, which is a fraction of the overall uncertainty. Herein, we calibrate a TWIMS instrument and derive TW CCS N2 and TW CCS N2→He values for four proteins: cytochrome c, ubiquitin, apo-myoglobin, and holo-myoglobin. We show that compared to reporting only the standard deviation of TW CCS, propagating error through the calibration results in a significant increase in the number of calculated TW CCS values that agree within experimental error with literature values ( DT CCS). Incorporating this additional uncertainty provides a more thorough assessment of a protein ion's gas-phase conformations, enabling the structures sampled by native IM-MS to be compared against other reported structures, both experimental and computational.

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Decoding Protein Gas‐Phase Stability with Alanine Scanning and Collision‐Induced Unfolding Ion Mobility Mass Spectrometry

Cited by 6 publications

References 52 publications

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility‐Mass Spectrometry Provides Insights into Protein Gas‐Phase Structure

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility‐Mass Spectrometry Provides Insights into Protein Gas‐Phase Structure

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Propagating Error through Traveling-Wave Ion Mobility Calibration

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