Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.     

Robust analysis of the yeast proteome under 50 kDa by molecular-mass-based fractionation and top-down mass spectrometry

As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0-50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC-MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins.     

Humic substances in soils: are they really chemically distinct?

Humic substances (HS) are an operationally defined fraction of soil organic matter, and they represent the largest pool of recalcitrant organic carbon in the terrestrial environment. It has traditionally been thought that extractable HS consist of novel categories of cross-linked macromolecular structures. In this study, advanced nuclear magnetic resonance approaches were used to study the major components (proteins, carbohydrates, aliphatic biopolymers, and lignin) that are known to be present in HS, and to identify their fingerprints in humic mixtures. Theoretically, once all known components have been identified, the remaining signals should be from materials with novel structures, themselves forming a distinct chemical category of humic materials. Surprisingly, nearly all of the NMR signals in traditional HS fractions could be assigned to intact and degrading biopolymers. We therefore suggest that the vast majority of operationally defined humic material in soils is a very complex mixture of microbial and plant biopolymers and their degradation products but not a distinct chemical category. It is important to note this work in no way rules out the existence of a distinct category of humic macromolecules, either at low abundance in the soluble fraction from young soils, in diagenetically evolved samples (for example lignites, etc.), or in the nonextractable humin fraction.     

Evidence for oligomer formation in clouds: reactions of isoprene oxidation products

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate product formation in laboratory experiments designed to study secondary organic aerosol (SOA) formation in clouds. It has been proposed that water soluble aldehydes derived from aromatics and alkenes, including isoprene, oxidize further in cloud droplets forming organic acids and, upon droplet evaporation, SOA. Pyruvic acid is an important aqueous-phase intermediate. Time series samples from photochemical batch aqueous phase reactions of pyruvic acid and hydrogen peroxide were analyzed for product formation. In addition to the monomers predicted by the reaction scheme, products consistent with an oligomer system were found when pyruvic acid and OH radical were both present. No evidence of oligomer formation was found in a standard mix composed of pyruvic, glyoxylic, and oxalic acids prepared in the same matrix as the samples analyzed using the same instrument conditions. The distribution of high molecular weight products is consistent with oligomers composed of the mono-, oxo-, and di-carboxylic acids expected from the proposed reaction scheme.     

Conversational Voice, Communicated Commitment, and Public Relations Outcomes in Interactive Online Communication

Organizations face unique challenges in communicating interactively online with publics that comprise dauntingly large numbers of individuals. This online survey examined the perceptions of people who had experienced interactive communication with a large consumer-tech-industry company via organizational blogs. Those reporting the greatest exposure to the blogs in this study were more likely to perceive the organization as communicating with a conversational voice. Conversational human voice and communicated relational commitment (relational maintenance strategies) correlated positively with trust, satisfaction, commitment, and control mutuality (relational outcomes). Building on prior research, this survey supports a model of distributed public relations—one in which key outcomes of public relations are fostered by a wide range of people communicating interactively while representing an organization.     

Atmospheric Trace Molecule Spectroscopy (ATMOS) Experiment Version 3 data retrievals

Version 3 of the Atmospheric Trace Molecule Spectroscopy (ATMOS) experiment data set for some 30 trace and minor gas profiles is available. From the IR solar-absorption spectra measured during four Space Shuttle missions (in 1985, 1992, 1993, and 1994), profiles from more than 350 occultations were retrieved from the upper troposphere to the lower mesosphere. Previous results were unreliable for tropospheric retrievals, but with a new global-fitting algorithm profiles are reliably returned down to altitudes as low as 6.5 km (clouds permitting) and include notably improved retrievals of H2O, CO, and other species. Results for stratospheric water are more consistent across the ATMOS spectral filters and do not indicate a net consumption of H2 in the upper stratosphere. A new sulfuric-acid aerosol product is described. An overview of ATMOS Version 3 processing is presented with a discussion of estimated uncertainties. Differences between these Version 3 and previously reported Version 2 ATMOS results are discussed. Retrievals are available at http://atmos.jpl.nasa.gov/atmos.     

The effect of protein profile and preheating on denaturation of whey proteins and development of viscosity in milk protein beverages during heat treatment

The effect of preheat temperature (63 or 77 °C for 30 s; final heat 120 °C for 30 s) and casein to whey protein ratio on the physical characteristics of 3.3%, w/w, dairy protein beverages was investigated. Dispersions preheated at 77 °C had lower viscosity than dispersions preheated at 63 °C. Casein‐containing dispersions had significantly lower levels of α‐lactalbumin denaturation than whey protein‐only dispersions. A higher proportion of casein improved the thermal stability of protein dispersions. Overall, alteration of preheat temperature and casein to whey protein ratio can influence dairy beverage quality, with increasing levels of casein reducing physical changes due to heat treatment.     

Fate of the fusarium mycotoxins,deoxynivalenol, nivalenol and zearalenone,during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.     

Integrated bulk/SOI APD sensor: bulk substrate inspection with Geiger-mode avalanche photodiodes

A rapid assessment of bulk silicon quality after removal of the silicon-on-insulator (SOI) and buried oxide layer using both plasma and wet etch is compared with standard p-epi silicon by comparing the performance of avalanche photodiodes (APD) operated in Geiger-mode. Plasma etching of the buried oxide shows lower dark counts than wet etched or standard p-epi substrates.     

Intracellular motility: how can we all work together?

Recent results reinforce the view that actin-based and microtubule-based motility systems do not operate independently, but are used in coordinated fashion to determine intracellular localization of cargo such as organelles.