Nonresonant femtosecond laser vaporization of aqueous protein preserves folded structure

Abstract

Femtosecond laser vaporization-based mass spectrometry can be used to measure protein conformation in vitro at atmospheric pressure. Cytochrome c and lysozyme are vaporized from the condensed phase into the gas phase intact when exposed to an intense (10 13 W∕cm 2 ), nonresonant (800 nm), ultrafast (75 fs) laser pulse. Electrospray postionization time-of-flight mass spectrometry reveals that the vaporized protein maintains the solution-phase conformation through measurement of the charge-state distribution and the collision-induced dissociation channels.multiphoton | nonthermal T he interaction of intense, ultrashort laser pulses with matter has resulted in a number of remarkable unique phenomena including above threshold ionization (1, 2), high harmonic generation (3-5), Coulomb explosion (6), nonadiabatic excitation of polyatomic molecules (7,8), neutron emission from clusters (9, 10), and the creation of attosecond laser pulses (5). Intense lasermatter interactions are currently used to determine polyatomic molecular electronic structure (11) and nuclear dynamics (12). Biomolecular structure determination represents a unique frontier for ultraintense laser experiments. In this regard, methods to deliver nonvolatile biological molecules intact into the gas phase, preferably maintaining condensed phase structure, are of interest. We recently reported that intense, nonresonant, ultrafast laser pulses could be used to deliver proteins with molecular weights up to 45 kDa into the gas phase without fragmentation (13). One motivation for this investigation is that for femtosecond laser vaporization to be of value for protein structural determination, the vaporization must ideally preserve the condensed phase primary, secondary, and tertiary conformation. Another motivation is the fact that the analysis of biological structure in situ presents a significant challenge. As a first step toward this goal, we investigate the conformation of proteins vaporized from the solution phase, in vitro, using intense, nonresonant femtosecond laser pulses.The biological function of a protein is determined by both primary sequence and structural conformation, with the latter governed by inter-and intramolecular covalent and noncovalent interactions. Techniques such as X-ray crystallography (14), NMR spectroscopy (15), and hydroxyl-radical protein footprinting (16) are commonly used to determine protein conformation and to probe noncovalent interactions. Another emerging method, electrospray ionization mass spectrometry (ESI-MS) (17) can be used to assess protein conformation in the electrospray solvent solution because folded protein typically displays one or two intense peaks in the mass spectrum (18). Protein in an unfolded conformation results in a bell-shaped distribution of the peaks with the centroid of the distribution occurring at a lower m∕z value (than the folded features) due to the basic amino acids being revealed and protonated (18). Changes in temperature, solvent, pH, and salt concentration will alter the equilibrium c...

Do Charge State Signatures Guarantee Protein Conformations?

“…The major advance claimed is that proteins vaporized by a femtosecond laser can retain a folded structure during this process. This observation is rationalized since the energy from the laser is deposited much faster than that required to produce unfolding or reorganization of protein structure [3]. The authors use the charge state signature of the protein to conclude that the folded conformation is retained during, and immediately after, laser desorption.…”

“…Levis and colleagues have described a femtosecond laser vaporization method in which folded protein structure is thought to be conserved [3]. In these experiments, a protein-containing solution, placed on a steel sample plate, is vaporized by a laser pulse.…”

“…This has come to the fore with several recent publications. The first publication involves an intriguing article in which the authors present evidence for preservation of folded structure following femtosecond laser vaporization with electrospray post-desorption [3]. The authors use the charge state signature of the protein to report on their conformation following laser vaporization, and compare the results of these experiments with 'conventional' electrospray.…”

Reply to Breuker et al.: How laser electrospray mass spectrometry (LEMS) measures condensed phase protein structure, not vacuum structure

“…2 arises because we defined gas phase as the transfer of protein into the atmosphere for subsequent entrainment into the electrospray ionization plume (1), and Breuker et al (2) define gas phase as the protein state after complete desolvation in vacuum. Our work showed that the measured charge state distribution detected in vacuum was a signature of the condensed phase structure (1).…”

“…We showed that the signatures for laser-vaporized protein revealed the anticipated charge state distribution, where conventional electrospray ionization (ESI) measurement promoted unfolding well before the vacuum state was obtained (1). Conventional ESI can distort protein structure determination because of both equilibrium ESI solvent effects and ionic effects in the Taylor cone.…”