[show abstract][hide abstract] ABSTRACT: Analysis of substrates directly on solid phase resins without the need for separate cleavage conditions remains an outstanding challenge in the field of solid phase synthesis. We now present the first example of simultaneous cleavage and mass spectrometric analysis of peptides from solid supports using direct analysis in real time (DART) mass spectrometry. We have shown that this method is compatible with a diverse array of solid phase resins and is suitable for analysis of both peptides and organic substrates.
[show abstract][hide abstract] ABSTRACT: Direct analysis in real time (DART) mass spectrometry is a recently developed innovative technology, which has shown broad applications for fast and convenient analysis of complex samples. Due to the ease of sample preparation, we have recently initiated an investigation of the feasibility of detecting nucleotides and nucleosides using the DART-AccuTOF instrument, which we will refer to as the DART mass spectrometer. Our experimental results reveal that the ions representing the intact molecules of nucleotides are not detectable in either positive-ion or negative-ion mode. Instead, all four natural nucleotides fragment in the DART ion source, and a common fragment ion, [C(5)H(5)O](+) (1), is observed, which is probably formed via multiple-elimination reactions. Interestingly, 1 can form adducts with nucleobases in different molar ratios in the DART ion source. In contrast to nucleotides, the ions representing the intact molecules of nucleosides are detected in both positive-ion and negative-ion mode using DART mass spectrometry. Surprisingly, the fragmentation pattern of nucleosides is different from that of nucleotides in the DART ion source. In the cases of nucleosides (under positive-ion conditions), the production of 1 is not observed, indicating that the phosphate group plays an important role for the multiple eliminations observed in the spectra of nucleotides. The in-source reactions described in the present work show the complexity of the conditions in the DART ion source, and we hope that our results illustrate a better understanding about DART mass spectrometry.
Journal of the American Society for Mass Spectrometry 04/2010; 21(8):1371-81. · 3.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Based on the concern about the presence of sulfur materials being in drywall (wallboard), a quick and reliable test to confirm the presence or absence of these materials using direct analysis in real time (DART) mass spectrometry in conjunction with an accurate-mass time-of-flight (TOF) mass spectrometer has been developed and is described here.
Journal of the American Society for Mass Spectrometry 08/2009; 20(11):2082-6. · 3.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: A method for analyzing pet food without sample processing is described for rapid identification of melamine based on mass spectrometry (MS) using soft ionization by direct analysis in real time (DART) to provide accurate measurement of mass and isotope-peak intensities, in-source collisionally activated dissociation (CAD) fragmentation, and determination of active hydrogens. Usually, MS analyses based on other than electron ionization (EI) spectra can be suspect because of the limited amount of information provided by a single mass spectral peak (or very few peaks). In such cases, additional degrees of confirmation are desirable to increase confidence in the experimental results. Chromatographic retention time can provide a degree of confidence; however, this requires time and, in some cases, detailed sample processing. Currently, the United States Food and Drug Administration uses a gas chromatography-EI-MS technique for the determination of melamine in pet food that involves sample extraction and derivatization prior to a lengthy chromatographic separation. In the method described here, identification is also confirmed through a determination of the number of active hydrogen atoms in the analyte molecule achieved by hydrogen/deuterium (H/D) exchange by treatment with deuterium oxide (D2O) at the initial stage of analysis. Cross-correlation of these four experimental data provides an unambiguous identification of melamine in contaminated pet food without the need for any sample preparation or chromatography. Limits of detection and the validity of the H/D exchange method as a confirmatory technique are also presented.
Journal of analytical toxicology 01/2007; 31(6):304-12. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.
Journal of Biological Chemistry 07/2005; 280(22):21220-30. · 4.65 Impact Factor