Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry.

In an effort to understand the role of molecular glycosylation in cancer a murine model has been used to characterize and fingerprint malignancies in established cell lines that manifest all the hallmarks of metastatic disease: spontaneous development, local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Using astrocyte cell controls, we compared N-linked glycosylation from a nonmetastatic brain tumor cell line and two different metastatic brain tumor cells. Selected ions in each profile were disassembled by ion trap mass spectrometry (MS(n)) which exhibited multiple structural differences between each tissue. These unique structures were identified within isomeric compositions as pendant nonreducing termini of di- and trisaccharide fragments, probably transparent to a tandem MS approach but distinctively not to sequential ion trap MS(n) detection.

Planning Military Drinking Water Needs: Development of a User-Friendly Smart Device Application.

Potable water is essential to maintain health and sustain military operations, but carrying and transporting water is a major logistical burden. Planning for group drinking water needs is complex, requiring understanding of sweat losses on the basis of intensity of activity, clothing biophysical parameters, and environmental conditions. Use of existing prediction equations is limited to tabled doctrine (e.g., Technical Bulletin, Medical 507) or to individuals with extensive expertise in thermal biophysics. In the present project, we translated the latest updated equations into a user-friendly Android application (Soldier Water Estimation Tool, SWET) that provides estimated drinking water required from 5 simple inputs based upon a detailed multiparametric sensitivity analysis. Users select from multiple choice inputs for activity level, clothing, and cloud cover, and manually enter exact values for temperature and relative humidity. Total drinking water needs for a unit are estimated in the Mission Planner tool on the basis of mission duration and number of personnel. In preliminary user acceptability testing, responses were overall positive in terms of ease of use and military relevance. Use of SWET for water planning will minimize excessive load (water) carriage in training and mission settings, and will reduce the potential for dehydration and/or hyponatremia to impair Warfighter health and performance.

The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS.

Thirteen high mannose isomers have been structurally characterized within three glycomers, Man(5)GlcNAc(2), Man(7)GlcNAc(2), and Man(8)GlcNAc(2) released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. Three previously characterized isomers from Man(7) and Man(8) (three each) have been identified plus one unreported Man(7) isomer. Incomplete alpha-glucosidase activity on the Man(6) and Man(7) glycoproteins appears to account for two additional isomeric structures. The preeminence of ion traps for detail analysis was further demonstrated by resolving three new isomers within the Man(5) glycomer summing to the six previously unreported structures in this glycoprotein. All reported structures represent a distribution of Golgi processing remnants that fall within the Man(9)GlcNAc(2) footprint. Topologies were defined by ion compositions along a disassembly pathway while linkage and branching were aided by spectral identity in a small oligomer fragment library. Isomers from this glycoprotein appear to represent a distribution of Golgi processing remnants, and an alphanumeric classification scheme has been devised to identify all products. Although numerous analytical strategies have been introduced to identify selected components of structure, it has been the continued focus of this and previous reports to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Duplication of these and results from comparable standards could bring an important analytical focus to carbohydrate sequencing that is greatly lacking.

Carbohydrate structural isomers analyzed by sequential mass spectrometry.

Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (MSn) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following MS2 and required multiple (MSn>2) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation "scars" throughout the MSn tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the MSn data sets.

Congruent strategies for carbohydrate sequencing. 3. OSCAR: an algorithm for assigning oligosaccharide topology from MSn data.

This is the third in a sequence of reports devoted to the development of congruent strategies for carbohydrate sequencing. Two previous reports outlined the strategies for observing structural detail from MSn data and introduced tools that compile, search, and compare fragment spectra in a bottom-up approach to oligosaccharide sequencing. In this third report, we introduce the operational details of an algorithm that we define as the Oligosaccharide Subtree Constraint Algorithm (OSCAR). This algorithm assimilates analyst-selected MSn ion fragmentation pathways into oligosaccharide topology (branching and linkage) using what may be considered a top-down sequencing strategy. Guided by a series of logical constraints, this de novo algorithm provides molecular topology without presumed biosynthetic constraints or external comparisons. In this introductory study, OSCAR is applied to a series of permethylated oligomers and isomeric glycans, and topologies are assigned in a few hundredths of a second.