An electron-ion coincidence spectrometer for commissioning of a synchrotron radiation beamline: Absolute photon intensity and content of higher harmonic radiation.

We describe the commissioning of a new electron-ion coincidence spectrometer used to diagnose the photon beam from a plane grating monochromator beamline at the ASTRID2 synchrotron radiation facility. The spectrometer allows determination of the absolute photon intensity by calibration to the photoabsorption cross sections of known gases, such as the rare gases Ar, Kr, and Xe presented here. The spectrometer operates at very low pressure (∼10-8-10-9 mbar) and-due to the coincidence electron-ion detection scheme-the detector efficiencies can be determined routinely; hence, the spectrometer can be recalibrated swiftly. By variation of a single potential of the spectrometer, the content of higher order radiation in the monochromatized synchrotron radiation can be analyzed. The layout and operation of the synchrotron radiation beamline at ASTRID2 and its additional photon diagnostic units are additionally described.

Origin of the Intrinsic Fluorescence of the Green Fluorescent Protein.

Green fluorescent protein, GFP, has revolutionized biology, due to its use in bioimaging. It is widely accepted that the protein environment makes its chromophore fluoresce, whereas the fluorescence is completely lost when the native chromophore is taken out of GFP. By the use of a new femtosecond pump-probe scheme, based on time-resolved action spectroscopy, we demonstrate that the isolated deprotonated GFP chromophore can be trapped in the first excited state when cooled to 100 K. The trapping is shown to last for 1.2 ns, which is long enough to establish conditions for fluorescence and consistent with calculated trapping barriers in the electronically excited state. Thus, GFP fluorescence is traced back to an intrinsic chromophore property, and by improving excited-state trapping, protein interactions enhance the molecular fluorescence.

A cylindrical quadrupole ion trap in combination with an electrospray ion source for gas-phase luminescence and absorption spectroscopy.

A relatively simple setup for collection and detection of light emitted from isolated photo-excited molecular ions has been constructed. It benefits from a high collection efficiency of photons, which is accomplished by using a cylindrical ion trap where one end-cap electrode is a mesh grid combined with an aspheric condenser lens. The geometry permits nearly 10% of the emitted light to be collected and, after transmission losses, approximately 5% to be delivered to the entrance of a grating spectrometer equipped with a detector array. The high collection efficiency enables the use of pulsed tunable lasers with low repetition rates (e.g., 20 Hz) instead of continuous wave (cw) lasers or very high repetition rate (e.g., MHz) lasers that are typically used as light sources for gas-phase fluorescence experiments on molecular ions. A hole has been drilled in the cylinder electrode so that a light pulse can interact with the ion cloud in the center of the trap. Simulations indicate that these modifications to the trap do not significantly affect the storage capability and the overall shape of the ion cloud. The overlap between the ion cloud and the laser light is basically 100%, and experimentally >50% of negatively charged chromophore ions are routinely photodepleted. The performance of the setup is illustrated based on fluorescence spectra of several laser dyes, and the quality of these spectra is comparable to those reported by other groups. Finally, by replacing the optical system with a channeltron detector, we demonstrate that the setup can also be used for gas-phase action spectroscopy where either depletion or fragmentation is monitored to provide an indirect measurement on the absorption spectrum of the ion.

Analysis of ionic photofragments stored in an electrostatic storage ring.

A new method to analyze the properties of fragment ions created in storage ring experiments is presented. The technique relies on an acceleration of ionic fragments immediately after production whereby the fragments are stored in the storage ring. To obtain a fragment mass spectrum, the storage ring is exploited as an electrostatic analyzer (ESA) in which case the number of stored fragment ions is recorded as a function of the applied acceleration potential. However, the storage ring can additionally be employed as a time-of-flight (TOF) instrument by registering the temporal distribution of fragment ions. It is demonstrated that the combined ESA-TOF operation of the ring allows not only to determine fragment masses with much better resolution compared to the ESA mode alone but also enables the extraction of detailed information on the fragmentation dynamics. The method is described analytically and verified with photodissociation experiments on stored Cl2 (-) at an excitation wavelength of 530 nm.

IR-induced conformational isomerization of a helical peptide in a cold ion trap.

In this work, we use laser-induced population transfer techniques to study the conformational isomerization of a helical peptide, Ac-Phe-(Ala)5-LysH(+), in a cold ion trap. In one scheme, called IR-UV hole-filling spectroscopy, a single conformation is selectively excited with an IR pump laser via a distinct NH stretch vibration. After giving the vibrationally excited ions sufficient time to isomerize and re-cool in the trap, the new conformational redistribution is detected by UV photofragment spectroscopy. While we clearly observe a redistribution of the conformer populations due to isomerization, only those conformations that initially have population participate in this redistribution-we do not form conformers that were not initially present in the trap. In a second scheme, called IR-induced population transfer spectroscopy, we determine the fractional populations of the four stable conformations of Ac-Phe-(Ala)5-LysH(+) by scanning the IR laser while selectively detecting a specific conformation using UV photofragment spectroscopy.

Conformational distribution of bradykinin [bk + 2 H]2+ revealed by cold ion spectroscopy coupled with FAIMS.

We employ cold ion spectroscopy (CIS) in conjunction with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to study the peptide bradykinin in its doubly protonated charge state ([bk + 2 H](2+)). Using FAIMS, we partially separate the electrosprayed [bk + 2 H](2+) ions into two conformational families and selectively introduce one of them at a time into a cold ion trap mass spectrometer, where we probe them by UV photofragment spectroscopy. Although the two conformational families have distinct electronic spectra, some cross-conformer contamination can be observed under certain conditions. We demonstrate that this contamination comes from isomerization of ions energized during and/or after their separation and not from incomplete separation of the initially electrosprayed conformations in the FAIMS stage. By varying the injection voltage of the ions into our mass spectrometer, we can intentionally induce isomerization to produce what seems to be a gas phase equilibrium distribution of conformers. This distribution is different from the one produced initially by electrospray, indicating that some of the conformers are kinetically trapped and may be related to conformers that are more favored in solution.

Spectroscopy of mobility-selected biomolecular ions.

We describe here experiments that combine differential ion mobility, which separates conformational isomers of biomolecular ions, with electronic spectroscopy in a cold, radio-frequency ion trap. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. We demonstrate here that using differential ion mobility to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techniques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasibility and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray.

A new tandem mass spectrometer for photofragment spectroscopy of cold, gas-phase molecular ions.

We present here the design of a new tandem mass spectrometer that combines an electrospray ion source with a cryogenically cooled ion trap for spectroscopic studies of cold, gas-phase ions. The ability to generate large ions in the gas phase without fragmentation, cool them to approximately 10 K in an ion trap, and perform photofragment spectroscopy opens up new possibilities for spectroscopic characterization of large biomolecular ions. The incorporation of an ion funnel, together with a number of small enhancements, significantly improves the sensitivity, signal stability, and ease of use compared with the previous instrument built in our laboratory.

Probing the sub-microsecond photodissociation dynamics in gas-phase retinal chromophores.

The photoinduced fragmentation of a retinal model chromophore (all-trans-n-butyl protonated Schiff-base retinal) was studied in vacuo using a new experimental technique. The apparatus is able to record the photodissociation yield of gas-phase biomolecular ions in the first microseconds after absorption. Together with the existing ion storage ring ELISA, which operates on the millisecond to second time scale, the complete decay dynamics of such molecules can now be followed. In the case of retinal, the time-dependent fragmentation yield observed after irradiation with a 410 nm laser pulse exhibits contributions from one- and two-photon absorption, which decay non-exponentially with lifetimes on the order of 1 ms and 1 micros, respectively. The decay can be simulated using a statistical model, yielding good agreement with the experimental findings on both the millisecond and the microsecond time scales. No indication for nonstatistical processes is found for this molecule, the upper limit for a possible direct rate being a factor of 10(4) below the observed statistical dissociation rate.

Time-resolved study of solvent-induced recombination in photodissociated IBr-(CO2)n clusters.

We report the time-resolved recombination of photodissociated IBr-(CO2)n (n = 5-10) clusters following excitation to the dissociative IBr-A' 2Pi12 state of the chromophore via a 180 fs, 795 nm laser pulse. Dissociation from the A' state of the bare anion results in I- and Br products. Upon solvation with CO2, the IBr- chromophore regains near-IR absorption only after recombination and vibrational relaxation on the ground electronic state. The recombination time was determined by using a delayed femtosecond probe laser, at the same wavelength as the pump, and detecting ionic photoproducts of the recombined IBr- cluster ions. In sharp contrast to previous studies involving solvated I2-, the observed recombination times for IBr-(CO2)n increase dramatically with increasing cluster size, from 12 ps for n = 5 to 900 ps for n = 8,10. The nanosecond recombination times are especially surprising in that the overall recombination probability for these cluster ions is unity. Over the range of 5-10 solvent molecules, calculations show that the solvent is very asymmetrically distributed, localized around the Br end of the IBr- chromophore. It is proposed that this asymmetric solvation delays the recombination of the dissociating IBr-, in part through a solvent-induced well in the A' state that (for n = 8,10) traps the evolving complex. Extensive electronic structure calculations and nonadiabatic molecular dynamics simulations provide a framework to understand this unexpected behavior.