A Workflow for Meaningful Interpretation of Classification Results from Handheld Ambient Mass Spectrometry Analysis Probes.

While untargeted analysis of biological tissues with ambient mass spectrometry analysis probes has been widely reported in the literature, there are currently no guidelines to standardize the workflows for the experimental design, creation, and validation of molecular models that are utilized in these methods to perform class predictions. By drawing parallels with hurdles that are faced in the field of food fraud detection with untargeted mass spectrometry, we provide a stepwise workflow for the creation, refinement, evaluation, and assessment of the robustness of molecular models, aimed at meaningful interpretation of mass spectrometry-based tissue classification results. We propose strategies to obtain a sufficient number of samples for the creation of molecular models and discuss the potential overfitting of data, emphasizing both the need for model validation using an independent cohort of test samples, as well as the use of a fully characterized feature-based approach that verifies the biological relevance of the features that are used to avoid false discoveries. We additionally highlight the need to treat molecular models as "dynamic" and "living" entities and to further refine them as new knowledge concerning disease pathways and classifier feature noise becomes apparent in large(r) population studies. Where appropriate, we have provided a discussion of the challenges that we faced in our development of a 10 s cancer classification method using picosecond infrared laser mass spectrometry (PIRL-MS) to facilitate clinical decision-making at the bedside.

Robust Fluorescence Collection Module for Wide-Bore Ion Cyclotron Resonance Mass Spectrometers.

Mass spectrometers are at the heart of the most powerful toolboxes available to scientists when studying molecular structure, conformation, and dynamics in controlled molecular environments. Improved molecular characterization brought about by the implementation of new orthogonal methods into mass spectrometry-enabled analyses opens deeper insight into the complex interplay of forces that underlie chemistry. Here, we detail how one can add fluorescence detection to commercial ultrahigh-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers without adverse effects to its preexisting analytical tools. This advance enables measurements based on fluorescence detection, such as Förster resonance energy transfer (FRET), to be used in conjunction with other MS/MS techniques to probe the conformation and dynamics of large biomolecules, such as proteins and their complexes, in the highly controlled environment of a Penning trap.

Dual Laser and Desorption Electrospray Ionization Mass Spectrometry Imaging Using the Same Interface.

For a more comprehensive characterization of molecular heterogeneities of matter, multimodal mass spectrometry imaging must be developed to take advantage of the complementarity of information available through different ionization mechanisms. We report the design, implementation, and performance validation of a laser desorption imaging interface composed of add-on components that adapt a commercial Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging interface for dual imaging of Picosecond Infrared Laser Mass Spectrometry (PIRL-MS) with DESI-MS. The interface utilizes hardware elements and data analysis pipelines already established for DESI-MS imaging, and was further validated in cancer margin assessments using human medulloblastoma cancers. The PIRL-MS images were robust and reproducible across multiple experimental runs on independently prepared xenograft tumors, and could be segmented into cancer and healthy regions in concordance with pathology using a variety of supervised and unsupervised clustering methods. The spectral quality and complexity obtained with this interface were examined with infiltrating and noninfiltrating tumors, and were comparable to other mass spectrometry analysis interfaces. The average PIRL-MS spectra from spatially resolved images could be used for robust cancer m/z model building to classify medulloblastoma cancer from healthy tissue without any misclassifications, an observation that held true over close to 70 sampling data points. While the unsupervised spectral analysis methods suggested a slight suppression of signal in the phospholipid range compared to the hand-held configuration, these changes were insufficient to hamper utility in cancer margin assessment with spatially resolved data obtained with our interface. Dual PIRL-MS and DESI-MS imaging of consecutive sections, as suggested by multivariate loading plots, revealed highly complementary molecular information with m/z values identifiable with one desorption method sufficient to reveal cancer regions being absent in another, further emphasizing the need for effective hardware and software interfaces for dual mass spectrometry imaging.

Ambient Mass Spectrometry Imaging with Picosecond Infrared Laser Ablation Electrospray Ionization (PIR-LAESI).

A picosecond infrared laser (PIRL) is capable of cutting through biological tissues in the absence of significant thermal damage. As such, PIRL is a standalone surgical scalpel with the added bonus of minimal postoperative scar tissue formation. In this work, a tandem of PIRL ablation with electrospray ionization (PIR-LAESI) mass spectrometry is demonstrated and characterized for tissue molecular imaging, with a limit of detection in the range of 100 nM for reserpine or better than 5 nM for verapamil in aqueous solution. We characterized PIRL crater size using agar films containing Rhodamine. PIR-LAESI offers a 20-30 µm vertical resolution (∼3 µm removal per pulse) and a lateral resolution of ∼100 µm. We were able to detect 25 fmol of Rhodamine in agar ablation experiments. PIR-LAESI was used to map the distribution of endogenous methoxykaempferol glucoronide in zebra plant (Aphelandra squarrosa) leaves producing a localization map that is corroborated by the literature. PIR-LAESI was further used to image the distribution inside mouse kidneys of gadoteridol, an exogenous magnetic resonance contrast agent intravenously injected. Parallel mass spectrometry imaging (MSI) using desorption electrospray ionization (DESI) and matrix assisted laser desorption ionization (MALDI) were performed to corroborate PIR-LAESI images of the exogenous agent. We further show that PIR-LAESI is capable of desorption ionization of proteins as well as phospholipids. This comparative study illustrates that PIR-LAESI is an ion source for ambient mass spectrometry applications. As such, a future PIRL scalpel combined with secondary ionization such as ESI and mass spectrometry has the potential to provide molecular feedback to guide PIRL surgery.

Fluorescence imaging for visualization of the ion cloud in a quadrupole ion trap mass spectrometer.

Laser-induced fluorescence is used to visualize populations of gaseous ions stored in a quadrupole ion trap (QIT) mass spectrometer. Presented images include the first fluorescence image of molecular ions collected under conditions typically used in mass spectrometry experiments. Under these "normal" mass spectrometry conditions, the radial (r) and axial (z) full-width at half maxima (FWHM) of the detected ion cloud are 615 and 214 µm, respectively, corresponding to ~6% of r0 and ~3% of z0 for the QIT used. The effects on the shape and size of the ion cloud caused by varying the pressure of helium bath gas, the number of trapped ions, and the Mathieu parameter q z are visualized and discussed. When a "tickle voltage" is applied to the exit end-cap electrode, as is done in collisionally activated dissociation, a significant elongation in the axial, but not the radial, dimension of the ion cloud is apparent. Finally, using spectroscopically distinguishable fluorophores of two different m/z values, images are presented that illustrate stratification of the ion cloud; ions of lower m/z (higher qz) are located in the center of the trapping region, effectively excluding higher m/z (lower qz) ions, which form a surrounding layer. Fluorescence images such as those presented here provide a useful reference for better understanding the collective behavior of ions in radio frequency (rf) trapping devices and how phenomena such as collisions and space-charge affect ion distribution.

Self-localizing stabilized mega-pixel picoliter arrays with size-exclusion sorting capabilities.

We report on a liquid self-localizing process capable of producing Mega-pixel arrays of picoliter volumes on a 1 cm(2) area, within seconds, for high throughput sampling. The chip is based on principles of spatially varying wetting and stabilization. The key is to develop differential surface contact regions, which lead to both localization of the solution and increasing the surface adsorption energy to further pin the liquid to the surface, as highlighted by other studies. By exploiting surface roughness for enhanced wettability, we demonstrate wetting of wells with the aspect ratio of 100. The high precision of reactive ion etching (RIE) of silicon substrates allows for an extremely reproducible method of preparing the array of identical well structures and increased contact area to increase surface adsorption in the wells. "Dynamic wetting" is then readily achieved through inducing contact line instability by simply moving a drop of liquid on the top surface of the array. Liquid samples self-localize into the array pattern with the associated liquid flow leading to self-localization of suspended particles or analyte. The resulting picoliter volumes are both spatially ordered and stable for long periods of time, even for such small volumes, to permit selective measurements of the contents. This development will be particularly important in the assembly of the massive amounts of crystalline particles needed for atomically resolved structural dynamics using the latest advances in high number density electron and X-ray sources.

Fluorescence resonance energy transfer in gaseous, mass-selected polyproline peptides.

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.

Gas-phase fluorescence excitation and emission spectroscopy of mass-selected trapped molecular ions.

A flexible interface to perform optical spectroscopic measurements on gaseous ions stored in a modified commercial quadrupole ion trap (QIT) mass spectrometer is described. The modifications made to the mass spectrometer did not adversely affect its operating characteristics. Gas-phase ions are produced using electrospray ionization, mass isolated and stored in the trapping mass spectrometer. The ions are subsequently irradiated with visible light from a tunable laser and dispersed fluorescence spectra are recorded simultaneously. Mass spectra are recorded after the irradiation period. This set-up allows us to track a range of possible outcomes upon photoexcitation of selected ions including fluorescence, photofragmentation and photodetachment of electrons. The experimental set-up is characterized using rhodamine 590, which is a methyl ester variant of rhodamine 6G. Fluorescence excitation and emission spectra of gaseous rhodamine 590 are measured and compared with solution-phase spectra. Excitation and emission maxima for the gaseous ions are found to lie at higher energy than for the solvated rhodamine 590. In addition, the gas-phase Stokes shift is significantly smaller than the solution-phase Stokes shift. The effects of several experimental parameters on the observed fluorescence signal are investigated, including laser power, relative number of ions, q(z) trapping parameter and buffer gas pressure. In addition to its use for the photophysical characterization of the intrinsic properties of ionic chromophores, this set-up may be used to investigate the properties of mass-selected, dye-labeled biomolecules, both alone and in well-defined complexes and clusters.

Excited states and energy transfer among DNA bases in double helices.

The study of excited states and energy transfer in DNA double helices has recently gained new interest connected to the development of computational techniques and that of femtosecond spectroscopy. The present article points out contentious questions regarding the nature of the excited states and the occurrence of energy transfer and shows how they are currently approached. Using as example the polymer poly(dA) . poly(dT), composed of about 2000 adenine-thymine pairs, a model is proposed on the basis of time-resolved measurements (fluorescence decays, fluorescence anisotropy decays and fluorescence spectra, obtained with femtosecond resolution), associated to steady-state spectra. According to this qualitative model, excitation at 267 nm populates excited states that are delocalized over a few bases (excitons). Ultrafast internal conversion directs the excited state population to the lower part of the exciton band giving rise to fluorescence. Questions needing further investigations, both theoretical and experimental, are underlined with particular emphasis on delicate points related to the complexity and the plasticity of these systems.

Carbohydrate amino acids: the intrinsic conformational preference for a beta-turn-type structure in a carbopeptoid building block.

Infrared ion-dip spectroscopy coupled with DFT and ab initio calculations are used to establish the intrinsic conformational preference of the basic structural unit of a peptide mimic, a cis-tetrahydrofuran-based "carbopeptoid" (amide-sugar-amide), isolated at low temperature in the gas phase. The carbopeptoid units form a beta-turn-type structure, stabilized by an intramolecular NH --> O=C hydrogen bond across the sugar ring, thus forming a 10-membered, C10 turn. Despite the clear preference for C10 beta-turn structures in the basic unit, however, the presence of multiple hydrogen-bond donating and accepting groups also generates a rich conformational landscape, and alternative structures may be populated in related molecules. Calculations on an extended, carbopeptoid dimer unit, which includes an alternating amide-sugar-amide-sugar-amide chain, identify conformers exhibiting alternative hydrogen-bonding arrangements that are somewhat more stable than the lowest-energy double beta-turn forming conformer.

Molecular spectroscopy: complexity of excited-state dynamics in DNA.

Absorption of ultraviolet light by DNA is known to lead to carcinogenic mutations, but the processes between photon absorption and the photochemical reactions are poorly understood. In their study of the excited-stated dynamics of model DNA helices using femtosecond transient absorption spectroscopy, Crespo-Hernández et al. observe that the picosecond component of the transient signals recorded for the adenine-thymine oligonucleotide (dA)18.(dT)18 is close to that for (dA)18, but quite different from that for (dAdT)9.(dAdT)9; from this observation, they conclude that excimer formation limits excitation energy to one strand at a time. Here we use time-resolved fluorescence spectroscopy to probe the excited-state dynamics, which reveals the complexity of these systems and indicates that the interpretation of Crespo-Hernández et al. is an oversimplification. We also comment on the pertinence of separating base stacking and base pairing in excited-state dynamics of double helices and question the authors' assignment of the long-lived signal component found for (dA)18.(dT)18 to adenine excimers.

One- and two-photon ionization of DNA single and double helices studied by laser flash photolysis at 266 nm.

The ionization of the DNA single and double helices (dA)20, (dT)20, (dAdT)10(dAdT)10 and (dA)20(dT)20, induced by nanosecond pulses at 266 nm, is studied by time-resolved absorption spectroscopy. The variation of the hydrated electron concentration with the absorbed laser intensity shows that, in addition to two-photon ionization, one-photon ionization takes place for (dAdT)10(dAdT)10, (dA)20(dT)20 and (dA)20 but not for (dT)20. The spectra of all adenine-containing oligomers at the microsecond time-scale correspond to the adenine deprotonated radical formed in concentrations comparable to that of the hydrated electron. The quantum yield for one-photon ionization of the oligomers (ca. 10(-3)) is higher by at least 1 order of magnitude than that of dAMP, showing clearly that organization of the bases in single and double helices leads to an important lowering of the ionization potential. The propensity of (dAdT)10(dAdT)10, containing alternating adenine-thymine sequences, to undergo one-photon ionization is lower than that of (dA)20(dT)20 and (dA)20, containing adenine runs. Pairing of the (dA)20 with the complementary strand leads to a decrease of quantum yield for one photon ionization by about a factor of 2.

Collective behavior of Franck-Condon excited states and energy transfer in DNA double helices.

Absorption of UV radiation by DNA bases is known to induce carcinogenic mutations. The lesion distribution depends on the sequence around the hotspots, suggesting cooperativity between bases. Here we show that such cooperativity may intervene at the very first step of a cascade of events by formation of Franck-Condon states delocalized over several bases and subsequent energy transfer faster than 100 fs. Our study focuses on the double helix poly(dA).poly(dT), whose fluorescence, induced by femtosecond pulses at 267 nm, is probed by the upconversion technique and time-correlated single photon counting, over a large time domain (100 fs to 100 ns). The time-resolved fluorescence decays and fluorescence anisotropy decays are discussed in relation with the steady-state absorption and fluorescence spectra in the frame of exciton theory.

Photodissociation spectroscopy of trapped protonated tryptophan.

We have coupled a quadrupole ion trap with a frequency doubled optical parametric oscillator laser. The photodissociation spectrum of the protonated tryptophan ion from 215 to 320 nm is reported. The yields of fragmentation on each mass channel as a function of the laser wavelength were obtained. We also report experiments involving multiple stages of laser induced dissociation and discuss possible structures for the fragmentation products.

Probing the glycosidic linkage: UV and IR ion-dip spectroscopy of a lactoside.

The beta(1-->4) glycosidic linkage found in lactose is a prevalent structural motif in many carbohydrates and glycoconjugates. Using UV and IR ion-dip spectroscopies to probe benzyl lactoside isolated in the gas phase, we find that the disaccharide unit adopts only a single, rigid structure. Its fully resolved infrared ion-dip spectrum is in excellent agreement with that of the global minimum structure computed ab initio. This has glycosidic torsion angles of phi(H) (H1-C1-O-C4') approximately 180 degrees and psi(H) (C1-O-C4'-H4') approximately 0 degrees which correspond to a rotation of approximately 150 degrees about the glycosidic bond compared to the accepted solution-phase conformation. We discuss the biological implications of this discovery and the generality of the strategies employed in making it.

Hydrogen bond vibrations of 2-aminopyridine.2-pyridone, a Watson-Crick analogue of adenine.uracil.

The 2-aminopyridine.2-pyridone complex (2AP.2PY) is linked by antiparallel N-H.O=C and N-H.N hydrogen bonds, providing a model for the Watson-Crick hydrogen bond configuration of the adenine-thymine and adenine-uracil nucleobase pairs. Mass-selected S(1) <--> S(0) vibronic spectra of the supersonically cooled 2AP.2PY base pair analogue were measured by laser resonant two-photon ionization and emission spectroscopies. The hydrogen bond vibrations nu(2) (buckle, out-of-plane) and the three in-plane vibrations nu(3) (opening), nu(5) (shear), and nu(6) (stretch) were observed in the S(0) and S(1) states, giving detailed information on the stretching and deformation force constants of the (amide)N-H.N(pyridine) and the (amino)N-H.O=C hydrogen bonds. Density functional calculations with the B3LYP functional and the 6-311++G(d,p) and 6-311++G(2d,2p) basis sets yield ground-state hydrogen bond frequencies in close agreement with experiment.