Mass Spectrometry of Laser-Initiated Carbene Reactions for Protein Topographic Analysis

Self 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.

Section: Observations and An Improved Configurationmentioning

confidence: 98%

mentioning

confidence: 99%

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Ziemianowicz

Bomgarden

Etienne

et al. 2017

Self Cite

“…Because the reactive carbenes are in the L* side chains, the expected covalent links are of the L*-side-chain or L*-backbone type, allowing each original peptide to undergo collision-induced backbone dissociation independently. Such fragmentations, when they occur in tandem, do not allow simple Bb/y^nomenclature as in other new methods where crosslinking was carried out in solution and the products underwent a single dissociation [13][14][15].…”

Section: Glllg + Gl*l*l*kmentioning

confidence: 99%

“…This side reaction provides an internal clock that limits the time scale for carbene-X-H bond interactions to within 10 -7 s. The photochemical crosslinking (Bstitching^) can be readily detected by mass spectrometry, and the position of the new covalent link can be located by tandem mass spectrometry sequencing of gas-phase adduct ions [7]. A substantial drawback of solution studies is the very low yields of crosslinked products that must be separated from the unmodified starting material and side products [7,[13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler

Shaffer

Andrikopoulos

Řezáč

et al. 2016

Abstract. Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N 2 from the L* diazirine rings underwent insertion into X−H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific 13 C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H) + ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H] + ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase.

“…As we were in the process of revising the present manuscript, a carbene labeling approach was published based on the amino acid 'photoleucine' aimed at analyzing protein topography [57]. 'Photoleucine' is a large, chiral, zwitterionic, water-soluble molecule.…”

Section: Sites Of Labeling Can Be Pinpointed To Single Amino Acid Resmentioning

confidence: 99%

Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

Gómez

Mundo

Craig

et al. 2011

Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH 2 ). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites.