Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant(J5) E.coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS 3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts

Rapid food product analysis by surface acoustic wave nebulization coupled mass spectrometry

Rapid food product analysis is of great interest for quality control and assurance during the production process. Conventional quality control protocols require time and labor intensive sample preparation for analysis by state-of-the-art analytical methods. To reduce overall cost and facilitate rapid qualitative assessments, food products need to be tested with minimal sample preparation. We present a novel and simple method for assessing food product compositions by mass spectrometry using a novel surface acoustic wave nebulization method. This method provides significant advantages over conventional methods requiring no pumps, capillaries, or additional chemicals to enhance ionization for mass spectrometric analysis. In addition, the surface acoustic wave nebulization - mass spectrometry method is ideal for rapid analysis and to investigate certain compounds by using the mass spectra as a type of species-specific fingerprint analysis. We present for the first time surface acoustic wave nebulization generated mass spectra of a variety of fermented food products from a small selection of vinegars, wines, and beers

Analysis of endogenous D-amino acid-containing peptides in Metazoa

Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use L-amino acids to make proteins, a group of D-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the D-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-L sequence. Because this L-to-D post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides

Data from: Spatial genetic structure in American black bears (Ursus americanus): female philopatry is variable and related to population history

Previously, American black bears (Ursus americanus) were thought to follow the pattern of female philopatry and male-biased dispersal. However, recent studies have identified deviations from this pattern. Such flexibility in dispersal patterns can allow individuals greater ability to acclimate to changing environments. We explored dispersal and spatial genetic relatedness patterns across ten black bear populations—including long established (historic), with known reproduction >50 years ago, and newly established (recent) populations, with reproduction recorded <50 years ago—in the Interior Highlands and Southern Appalachian Mountains, United States. We used spatially-explicit, individual-based genetic simulations to model gene flow under scenarios with varying levels of population density, genetic diversity, and female philopatry. Using measures of genetic distance and spatial autocorrelation, we compared metrics between sexes, between population types (historic and recent), and among simulated scenarios which varied in density, genetic diversity, and sex-biased philopatry. In empirical populations, females in recent populations exhibited stronger patterns of isolation-by-distance (IBD) than females and males in historic populations. In simulated populations, low density populations had a stronger indication of IBD than medium to high density populations; however, this effect varied in empirical populations. Condition dependent dispersal strategies may permit species to cope with novel conditions and rapidly expand populations. Pattern-process modelling can provide qualitative and quantitative means to explore variable dispersal patterns, and could be employed in other species, particularly to anticipate range shifts in response to changing climate and habitat conditions