Scott C. Russell

Microorganism characterization by single particle mass spectrometry

In recent years a major effort by several groups has been undertaken to identify bacteria by mass spectrometry at the single cell level. The intent of this review is to highlight the recent progress made in the application of single particle mass spectrometry to the analysis of microorganisms. A large portion of the review highlights improvements in the ionization and mass analysis of bio-aerosols, or particles that contain biologically relevant molecules such as peptides or proteins. While these are not direct applications to bacteria, the results have been central to a progression toward single cell mass spectrometry. Developments in single particle matrix-assisted laser desorption/ionization (MALDI) are summarized. Recent applications of aerosol laser desorption/ionization (LDI) to the analysis of single microorganisms are highlighted. Successful applications of off-line and on-the-fly aerosol MALDI to microorganism detection are discussed. Limitations to current approaches and necessary future achievements are also addressed.

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ∼30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.

Targeted proteomics approach to species-level identification of Bacillus thuringiensis spores by AP-MALDI-MS

Anthrax infections progress at a rapid pace, making rapid detection methods of utmost importance. MALDI-MS proteomics methods focused on Bacillus anthracis detection have targeted chromosomally encoded proteins, which are highly conserved between closely related species, hindering species identification. Presented here is an AP-MALDI-MS method targeting plasmid-borne proteins from Bacillus spores for species-level identification. A bioinformatics analysis revealed that 60.3% and 75.4% of tryptic peptides from plasmid-borne proteins of B. anthracis and B. thuringiensis were species-specific, respectively. Reported here is a method in which plasmid-borne Δ-endotoxins were extracted directly from B. thuringiensis spores in 100 mM KOH. The pH was then adjusted to 8 and a 5-min trypsin digestion was performed on the extracted proteins. The resulting tryptic peptides were analyzed by AP-MALDI-MS/MS, which produced a definitive identification the B. thuringiensis speciesspecific Cry1Ab protein with a MASCOT score of 278 and expect value of 7.5 × 10−23. This method has demonstrated the detection and identification of B. thuringiensis spores at the species level following a 5-min trypsin digestion. The challenges in applying a similar approach to the detection of plasmid-borne protein toxins from B. anthracis are also discussed.

Near infrared reflectance spectroscopy (NIRS) analyses of nutrient composition and condensed tannin concentrations in carolina willow (Salix caroliniana)

Iron overload disorder has been described in a number of zoo-managed species, and it has been recommended to increase the tannin composition of the diet as a safe way to minimize iron absorption in these iron-sensitive species. The goal of this study was to examine the potential of near infrared reflectance spectroscopy (NIRS) as a rapid and simple screening tool to assess willow (Salix caroliniana) nutrient composition (crude protein: CP; acid detergent fiber: ADF; neutral detergent fiber: NDF; lignin, gross energy: GE) and condensed tannin (CT) concentrations. Calibration equations were developed by regression of the lab values from 2 years using partial least squares on n = 144 NIRS spectra to predict n = 20 independent validation samples. Using the full 2-year dataset, good prediction statistics were obtained for CP, ADF, NDF, and GE in plant leaves and stems (r(2 ) > 0.75). NIRS did not predict lignin concentrations reliably (leaves r(2)  = 0.52, stems r(2)  = 0.33); however, CTs were predicted moderately well (leaves r(2)  = 0.72, stems r(2)  = 0.67). These data indicate that NIRS can be used to quantify several key nutrients in willow leaves and stems including concentrations of plant secondary compounds which, depending on the bioactivity of the compound, may be targeted to feed iron-sensitive browsing animals.

Nutrient and plant secondary compound composition and iron-binding capacity in leaves and green stems of commonly used plant browse (Carolina willow; Salix caroliniana) fed to zoo-managed browsing herbivores.

Plant secondary compounds are diverse structurally, and associated biological effects can vary depending on multiple factors including chemical structure and reaction conditions. Phenolic compounds such as tannins can chelate dietary iron, and supplementation of animal species sensitive to iron overload with tannins may prevent/treat iron overload disorder. We assessed the nutrient and phenolic composition and iron-binding capacity of Carolina willow (Salix caroliniana), a plant fed to zoo-managed browsing herbivores. Based on studies in other plant species and the chemical structures of phenolic compounds, we hypothesized that the concentration of condensed tannins in willow would be inversely related to the concentration of phenolic glycosides and directly related to iron-binding capacity. Our results indicated that willow nutrient composition varied by year, season, and plant part, which could be taken into consideration when formulating animal diets. We also found that the predominant plant secondary compounds were condensed tannins with minimal phenolic glycosides. Instead of binding to iron, the willow leaf extracts reduced iron from the ferric to ferrous form, which may have prooxidative effects and increase the bioavailability of iron depending on animal species, gastrointestinal conditions, and whole animal processes. We recommend identifying alternative compounds that effectively chelate iron in vitro and conducting chelation therapy trials in vivo to assess potential effects on iron balance and overall animal health.

Programmed Assembly of Nanoscale Structures using Peptoids

Sequence-specific polymers are the basis of the most promising approaches to bottom-up programmed assembly of nanoscale materials. Examples include artificial peptides and nucleic acids. Another class is oligo(N-functional glycine)s, also known as peptoids, which permit greater sidegroup diversity and conformational control, and can be easier to synthesize and purify. We have developed a set of peptoids that can be used to make inorganic nanoparticles more compatible with biological sequence-specific polymers so that they can be incorporated into nucleic acid or other biologically based nanostructures. Peptoids offer degrees of modularity, versatility, and predictability that equal or exceed other sequence-specific polymers, allowing for rational design of oligomers for a specific purpose. This degree of control will be essential to the development of arbitrarily designed nanoscale structures.