Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly contributes to the outer part of the massive base. Quantitative evaluation of successive crossover spacings along individual F-actin filaments revealed the deviations from the mean repeat to be compensatory, i.e., short crossovers frequently followed long ones and vice versa. The variable crossover spacings and diameter of the F-actin filament together with the local unraveling of the two long-pitch helical strands are explained in terms of varying amounts of compensatory "lateral slipping" of the two strands past each other roughly perpendicular to the filament axis. This intrinsic disorder of the actin filament may enable the actin moiety to play a more active role in actin-myosin-based force generation than merely act as a rigid passive cable as has hitherto been assumed.     

Balanced macromolecular biosynthesis in "protoplasts" of Streptococcus faecalis

Osmotically fragile forms of Streptococcus faecalis 9790 were grown in 0.5 m sucrose- or 0.5 m NH(4)Cl-stabilized medium. The "protoplast" cultures exhibit an average growth rate constant of 0.66 to 0.94 mass doublings/hr. In a variety of experiments, turbidity and the net content of protein, ribonucleic acid (RNA) and deoxyribonucleic acid increase at the same rate, indicating balanced macromolecular biosynthesis. A total of two to three mass doublings was obtained, with no evidence of cell division. After osmotic shock, "protoplast" cultures released 93 to 94% of their RNA content in a form not sedimentable at 12,800 x g for 15 min, in contrast to streptococci, which released 7% of their RNA content after the same treatment.     

"Glyco-peakfinder"--de novo composition analysis of glycoconjugates

Mass spectrometric techniques are the key technology for rapid and reliable glycan analysis. However, the lack of robust, dependable, and freely available software for the (semi-) automatic annotation of mass spectra is still a severe bottleneck that hampers their rapid interpretation. In this article the "Glyco-Peakfinder" web-service is described allowing de novo determination of glycan compositions from their mass signals. Starting from a basic set of mandatory masses of glycan components, the calculation can be performed without any knowledge concerning the biological background of the sample or the fragmentation technique used. "Glyco-Peakfinder" assigns all types of fragment ions including monosaccharide cross-ring cleavage products and multiply charged ions. It provides full user control to handle modified glycans (persubstituted molecules, reducing-end modifications, glycoconjugates) and ion types. The formula applied to calculate the fragment masses and an outline of the implemented algorithm are discussed. A systematic evaluation of the dependence of all factors influencing the computation time revealed strikingly different impact of the individual calculation steps. To provide access to known carbohydrate structures a "composition search" in the open access database GLYCOSCIENCES.de can be performed. The service is available at the URL: www.eurocarbdb.org/applications/ms-tools.     

Characterization of the agent of "high plains disease": mass spectrometry determines the sequence of the disease-specific protein

The "32-kDa" protein specifically associated with high plains disease was characterized by time-of-flight mass spectrometry, after the agent had been isolated in pure culture by "vascular puncture inoculation," a novel mechanical means of transmission. Two isolates from different geographic locations each consisted of a mixture of subpopulations that were highly homologous to an amino acid sequence derived from a nucleotide sequence (U60141) deposited in GenBank trade mark by the Nebraska group as "the probable N-protein of high plains virus." However, the U60141 sequence was found to be incomplete; de novo sequencing of peptides produced by proteolytic digestions of the 32-kDa band from an SDS-PAGE separation showed that an additional 18 amino acid residues were present at the N terminus. BLAST (basic local alignment search tool) examination of the sequence showed no significant homology with any protein in the databases, indicating that the infectious agent of high plains disease is likely a member of a hitherto unclassified virus group.     

Mass spectrometry reveals that the antibiotic simocyclinone D8 binds to DNA gyrase in a "bent-over" conformation: evidence of positive cooperativity in binding

DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.     

A nonluminescent and highly virulent Vibrio harveyi strain is associated with "bacterial white tail disease" of Litopenaeus vannamei shrimp

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by "white tail" and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of "white tail" but of non-bacterial origin, the present disease was named as "bacterial white tail disease (BWTD)". Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.     

The track structure of alpha-particles from the point of view of ionization-cluster formation in "nanometric" volumes of nitrogen

Probability distributions of the size of ion clusters created in "nanometric" volumes of nitrogen by single alpha-particles of a gold-plated 241Am source, were measured and compared with those calculated by Monte Carlo methods in the same geometry. The diameter of the sensitive volumes had a mass per area of between 0.015 microgram/cm2 and 1.3 micrograms/cm2 which, for a material at unit density, corresponds to a nanometric target volume 0.15-13 nm in diameter. These nanometre sizes were simulated experimentally in a device called the Jet Counter. This consists of a pulse-operated valve which injects into an interaction chamber an expansion jet of molecular nitrogen gas, which is crossed by a narrow beam of alpha-particles. The resulting ions are counted and analyzed from the point of view of ionization cluster formation. The measured or calculated cluster size probabilities prove that the formation of ionization clusters along a "nanometre" track is governed by Poisson's law only in the case of very small target volumes, due to the contributions by secondary electrons. The present ionization cluster probabilities produced in "nanometric" volumes 0.15-13 nm in diameter, are the first ever determined experimentally and confirmed by Monte Carlo simulation.     

Body mass and foraging ecology predict evolutionary patterns of skeletal pneumaticity in the diverse "waterbird" clade

Extensive skeletal pneumaticity (air-filled bone) is a distinguishing feature of birds. The proportion of the skeleton that is pneumatized varies considerably among the >10,000 living species, with notable patterns including increases in larger bodied forms, and reductions in birds employing underwater pursuit diving as a foraging strategy. I assess the relationship between skeletal pneumaticity and body mass and foraging ecology, using a dataset of the diverse "waterbird" clade that encompasses a broad range of trait variation. Inferred changes in pneumaticity and body mass are congruent across different estimates of phylogeny, whereas pursuit diving has evolved independently between two and five times. Phylogenetic regressions detected positive relationships between body mass and pneumaticity, and negative relationships between pursuit diving and pneumaticity, whether independent variables are considered in isolation or jointly. Results are generally consistent across different estimates of topology and branch lengths. "Predictive" analyses reveal that several pursuit divers (loons, penguins, cormorants, darters) are significantly apneumatic compared to their relatives, and provide an example of how phylogenetic information can increase the statistical power to detect taxa that depart from established trait correlations. These findings provide the strongest quantitative comparative support yet for classical hypotheses regarding the evolution of avian skeletal pneumaticity.     

Rapid discrimination of Bacillus anthracis from other members of the B. cereus group by mass and sequence of "intact" small acid soluble proteins (SASPs) using mass spectrometry

The intentional contamination of buildings, e.g. anthrax in the bioterrorism attacks of 2001, demonstrated that the population can be affected rapidly and lethally if the appropriate treatment is not provided at the right time. Molecular approaches, primarily involving PCR, have proved useful in characterizing "white powders" used in these attacks as well as isolated organisms. However there is a need for a simpler approach, which does not involve temperamental reagents (e.g. enzymes and primers) which could potentially be used by first responders. It is demonstrated here that small acid-soluble proteins (SASPs), located in the core region of Bacillus spores, are reliable biomarkers for identification. The general strategy used in this study was to measure the molecular weight (MW) of an intact SASP by electrospray ionization mass spectrometry (ESI MS) followed by generation of sequence-specific information by ESI MS/MS (tandem mass spectrometry). A prominent SASP of mass 6679 was present in all B. anthracis strains. For B. cereus and B. thuringiensis strains the SASP had a mass of 6712. This represents a two amino acid substitution (serine to alanine; phenylalanine to tyrosine). The only SASP present in the B. anthracis genome consistent with this sequence is encoded by the gene ssB. This protein has a predicted mass of 6810, presumably post-translational processing leads to loss of methionine (mass 131) generating a SASP of mass 6679. This study showed that intact SASPs can be used as a biomarker for identification of B. anthracis; the protocol is simple and rapid. Extrapolation of this approach might prove important for real-time biodetection.     

Human body mass estimation: a comparison of "morphometric" and "mechanical" methods

In the past, body mass was reconstructed from hominin skeletal remains using both "mechanical" methods which rely on the support of body mass by weight-bearing skeletal elements, and "morphometric" methods which reconstruct body mass through direct assessment of body size and shape. A previous comparison of two such techniques, using femoral head breadth (mechanical) and stature and bi-iliac breadth (morphometric), indicated a good general correspondence between them (Ruff et al. [1997] Nature 387:173-176). However, the two techniques were never systematically compared across a large group of modern humans of diverse body form. This study incorporates skeletal measures taken from 1,173 Holocene adult individuals, representing diverse geographic origins, body sizes, and body shapes. Femoral head breadth, bi-iliac breadth (after pelvic rearticulation), and long bone lengths were measured on each individual. Statures were estimated from long bone lengths using appropriate reference samples. Body masses were calculated using three available femoral head breadth (FH) formulae and the stature/bi-iliac breadth (STBIB) formula, and compared. All methods yielded similar results. Correlations between FH estimates and STBIB estimates are 0.74-0.81. Slight differences in results between the three FH estimates can be attributed to sampling differences in the original reference samples, and in particular, the body-size ranges included in those samples. There is no evidence for systematic differences in results due to differences in body proportions. Since the STBIB method was validated on other samples, and the FH methods produced similar estimates, this argues that either may be applied to skeletal remains with some confidence.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

On the mechanism of reactions of nitrosoarenes with thiols. Formation of a common intermediate "semimercaptal"

To get more insight into the reactions of nitrosoarenes with thiols which may be responsible for cytotoxic effects, the reaction mechanism was studied with nitrosobenzene and 1-thioglycerol as model compounds. A transient intermediate was isolated by high performance liquid chromatography and identified as 2,3-dihydroxypropanesulfenic N-hydroxyphenylamide ("semimercaptal") by UV, 13C-NMR, and FAB mass spectroscopy. In aqueous solution this labile compound reassembles into 2,3-dihydroxypropanesulfinic phenylamide in a first order reaction. In the presence of excess thiol or ascorbic acid the "semimercaptal" is reduced to 2,3-dihydroxypropanesulfenic phenylamide without transient formation of a complete "mercaptal". Hydrolysis rather than thiolysis liberates aniline from the sulfenic phenylamide. Both the sulfinic and sulfenic phenylamides were obtained in crystalline form and identified by NMR and FAB mass spectroscopy. A scheme is presented of the known reactions of nitrosoarenes with thiols.     

The 19 S multicatalytic "prosome" proteinase is a constitutive enzyme in HeLa cells

HeLa human carcinoma cells contain a high molecular mass (Mr approximately 700,000) proteinase that is identical with the ubiquitous eukaryotic 19 S cylindrical "prosome" particle. Prosomes constitute 0.6% of cell bulk protein as determined by immunochemical analysis. The prosome proteinase is not a heat shock protein, nor is it induced by cell crowding in culture or by serum deprivation. Pulse-chase experiments showed that the proteinase is relatively stable in the cells (t 1/2 approximately 5 days).     

Revisited BIA-MS combination: entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.     

Nondestructive quantification of neutral lipids by thin-layer chromatography and laser-fluorescent scanning: suitable methods for "lipidome" analysis

A simple method for separation and quantification of neutral lipids was developed using thin-layer chromatography (TLC) and high-performance fluorescent scanning. Neutral lipid classes were separated using the double-developing TLC method and detected by rhodamine 6G and a laser-excited fluorescent scanner. The amount of lipids applied correlated with scanned intensity volume in a dose-dependent manner. The mass of each neutral lipid band was determined by comparing band intensities of unknown samples to dilution curves of authentic standards. After scanning the dye-sprayed TLC, acyl chain species of triglyceride (TG) extracted from TLC could be determined by gas chromatography. Using this method, we quantified the amounts of TG in mouse liver and found that the measured total mass of TG correlated with that obtained by enzymatic methods. Our method should provide the basic technique for "lipidome" analysis, designed to determine and compare total lipid classes and mass present in biological samples.