Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Vahidi, Siavash; Konermann, Lars

doi:10.1007/s13361-016-1389-x

Cited by 45 publications

(50 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, a recent analysis of reaction kinetics shows that radicals can persist for tens of milliseconds after initiation. Attempts to scavenge persistent radicals with glutamine was found to generate metastable secondary radicals that can further extend oxidation reactions [20].…”

mentioning

confidence: 99%

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Ziemianowicz

Bomgarden

Etienne

et al. 2017

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Abstract. Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS 2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments.

show abstract

mentioning

confidence: 99%

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Ziemianowicz

Bomgarden

Etienne

et al. 2017

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…Irradiation at 248 nm with a pulsed laser leads to the generation of Å OH that can react with solvent accessible side-chains [209]. The presence of scavengers (glutamine or histidine) tunes the lifetime of the Å OH such that a majority of the labelling reactions occur on timescales faster than protein folding/unfolding ($1 ls) [209][210][211], although recent evidence suggests that radicals may be longer-lived [212]. Alternatively, the scavenger dose can be tuned to extend/reduce the labelling pulse, allowing dose-response experiments to be performed [213].…”

Section: Hydroxyl Radical Footprintingmentioning

confidence: 99%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

View full text Add to dashboard Cite

a b s t r a c tThe last $25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.

show abstract

“…samples are exposed to a sub-microsecond burst of hydroxyl radicals generated by the laserinduced photolysis of hydrogen peroxide in a process termed Fast Photochemical Oxidation of Proteins (FPOP) 19 . The hydroxyl radicals are consumed faster than large conformational changes occur, with the vast majority of the radical reacted in under one microsecond 20,21 , although resulting protein-centered radicals are longer-lived 20,22 . Hydroxyl radicals oxidize amino acid side chains available on the surface in molecules in both conformation A and conformation B, with each side chain having a rate constant that is dependent upon the accessibility of that side chain in that protein conformation to the radical.…”

Section: Introductionmentioning

confidence: 99%

Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins Allows for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures

Tadi

Sharp

2019

Preprint

View full text Add to dashboard Cite

Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology, resulting in complex challenges in data analysis or complete failure of the method. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity will result in data reflecting a weighted average of all conformers, greatly complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size exclusion chromatography in an HPLC format with Hydroxyl Radical Protein Footprinting (HRPF) using online Fast Photochemical Oxidation of Proteins (FPOP). Using controlled mixtures of myoglobin and apomyoglobin as a model system to allow for controllable conformational heterogeneity, we demonstrate that we can obtain HRPF footprints of both holomyoglobin and apomyoglobin as they elute off of the SEC column.Comparison of online SEC-FPOP data of both mixture components with traditional FPOP data of each individual component shows that we can obtain the exact same footprinting pattern for each conformation in an online format with real-time FPOP. Using this method, conformations within conformationally heterogeneous mixtures can now be individually probed by SEC-FPOP, and the stability of the FPOP label allows this structural information to be retained.

show abstract

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Cited by 45 publications

References 65 publications

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Mass spectrometry-enabled structural biology of membrane proteins

Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins Allows for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures

Contact Info

Product

Resources

About