RESUMO
This work presents a benchtop method for collecting the room temperature gas phase infrared (IR) action spectra of protonated amino acids and their isomers. The adopted setup uses a minimally modified commercial electrospray ionization linear ion trap mass spectrometer (ESI-LIT-MS) coupled to a broadband continuous wave (cw) quantum cascade laser (QCL) source. This approach leverages messenger assisted action spectroscopic techniques using water-tagged molecular ions with complex formation, irradiation, and subsequent analysis, all taking place within a single linear ion trap stage. This configuration thus circumvents the use of multiple mass selection and analysis stages, cryogenic buffer cells, and complex high-power laser systems typically called upon to execute these techniques. The benchtop action spectrometer is used to collect the 935-1600 cm-1 (6.2-10.7 µm) IR action spectrum of a collection of amino acids and a dipeptide with results cross referenced against literature examples obtained with a free electron laser source. Recorded IR spectra are used for the analysis of binary mixture samples composed of constitutional isomers α-alanine and ß-alanine with ratios determined to â¼4% measurement uncertainty without the aid of a front-end separation stage. This turn-key QCL-based approach is a major step in showing the viability of tag-based action spectroscopic techniques for use in future in situ planetary science sensors and general analytical applications.
RESUMO
We report a spectrometer employing optically switched dual-wavelength cavity ring-down spectroscopy (OSDW-CRDS) for high-precision measurements of methane isotope ratios. A waveguide optical switch rapidly alternated between two wavelengths to detect absorption by two isotopologues using near-infrared CRDS. This approach alleviated common-mode noise that originated primarily from temperature and frequency fluctuations. We demonstrated the measurement of δD in natural abundance methane to a precision of 2.3 , despite the lack of active temperature or frequency stabilization of the cavity. The ability of alternating OSDW-CRDS to improve the isotope precision in the absence of cavity stabilization were measured by comparing the Allan deviation with that obtained when frequency-stabilizing the cavity length. The system can be extended to a wide variety of applications such as isotope analysis of other species, kinetic isotope effects, ortho-para ratio measurements, and isomer abundance measurements. Furthermore, our technique can be extended to multiple isotope analysis or two species involved in kinetics studies through the use of multiport or high-speed optical switches, respectively.
RESUMO
Recognition of DNA by proteins depends on DNA sequence and structure. Often unanswered is whether the structure of naked DNA persists in a protein-DNA complex, or whether protein binding changes DNA shape. While X-ray structures of protein-DNA complexes are numerous, the structure of naked cognate DNA is seldom available experimentally. We present here an experimental and computational analysis pipeline that uses hydroxyl radical cleavage to map, at single-nucleotide resolution, DNA minor groove width, a recognition feature widely exploited by proteins. For 11 protein-DNA complexes, we compared experimental maps of naked DNA minor groove width with minor groove width measured from X-ray co-crystal structures. Seven sites had similar minor groove widths as naked DNA and when bound to protein. For four sites, part of the DNA in the complex had the same structure as naked DNA, and part changed structure upon protein binding. We compared the experimental map with minor groove patterns of DNA predicted by two computational approaches, DNAshape and ORChID2, and found good but not perfect concordance with both. This experimental approach will be useful in mapping structures of DNA sequences for which high-resolution structural data are unavailable. This approach allows probing of protein family-dependent readout mechanisms.