Multipole-storage-assisted dissociation for the characterization of large proteins and simple protein mixtures by ESI-FTICR-MS.

In Fourier transform ion cyclotron resonance mass spectrometry, collisionally activated dissociation (CAD) typically is accomplished within the analyzer ion cell. An alternative approach of multipole-storage-assisted dissociation (MSAD) has previously been demonstrated by inducing collisional fragmentation in the external multipole that is usually employed for ion accumulation. To explore the utility of MSAD for interrogating intact proteins and simple protein mixtures in a multiplexed manner, we have investigated the means of controlling the collisional energy and the fragmentation pattern for this experimental approach.

Community proteomics of a natural microbial biofilm.

Using genomic and mass spectrometry–based proteomic methods, we evaluated gene expression, identified key activities, and examined partitioning of metabolic functions in a natural acid mine drainage (AMD) microbial biofilm community. We detected 2033 proteins from the five most abundant species in the biofilm, including 48% of the predicted proteins from the dominant biofilm organism, group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, which suggests that damage to biomolecules is a key challenge for survival.

Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure.

Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server.

DBDigger: reorganized proteomic database identification that improves flexibility and speed.

Database search identification algorithms, such as Sequest and Mascot, constitute powerful enablers for proteomic tandem mass spectrometry. We introduce DBDigger, an algorithm that reorganizes the database identification process to remove a problematic bottleneck. Typically such algorithms determine which candidate sequences can be compared to each spectrum.

Transcriptomic and proteomic characterization of the Fur modulon in the metal-reducing bacterium Shewanella oneidensis.

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon.

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.

A graph-theoretic approach for the separation of b and y ions in tandem mass spectra.

Motivation: Ion-type identification is a fundamental problem in computational proteomics. Methods for accurate identification of ion types provide the basis for many mass spectrometry data interpretation problems, including (a) de novo sequencing, (b) identification of post-translational modifications and mutations and (c) validation of database search results.

Results: Here, we present a novel graph-theoretic approach for solving the problem of separating b ions from y ions in a set of tandem mass spectra.

Comparison of sustained off-resonance irradiation collisionally activated dissociation and multipole storage-assisted dissociation for top-down protein analysis.

Tandem mass spectrometric data acquired for small (8-18 kDa) intact proteins by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) and multipole storage-assisted dissociation (MSAD) were compared, and the results indicate that the two activation methods do not always provide the same fragmentation patterns. In MSAD experiments, the charge state distribution made available by the ionization conditions may dictate the range of fragment ions that can be generated. In addition, conditions of high space charge within the hexapole impair transmission and/or trapping of high m/z species, which can result in loss of important precursor and product ions.

A computational method for assessing peptide- identification reliability in tandem mass spectrometry analysis with SEQUEST.

High-throughput protein identification in mass spectrometry is predominantly achieved by first identifying tryptic peptides by a database search and then by combining the peptide hits for protein identification. One of the popular tools used for the database search is SEQUEST. Peptide identification is carried out by selecting SEQUEST hits above a specified threshold, the value of which is typically chosen empirically in an attempt to separate true identifications from false ones.

New technologies for nutrition research.

The Experimental Biology 2003 symposium entitled "New Technologies for Nutrition Research" was organized to highlight new and emerging technologies, including nanotechnology and proteomics, and to suggest ways for their integration into nutrition research. Speakers focused on topics that included accelerator mass spectrometry for ultra-low level radiolabel tracing, nanodevices for real-time optical intracellular sensing, mass spectrometric techniques for examining protein expression, as well as potential applications for nanotechnology in the food sciences. These technologies may be particularly useful in obtaining accurate spatial information and low-level detection of essential and nonessential bioactive food components (nutrients) and their metabolites, and in enhancing the understanding of the impact of nutrient/metabolite and biomolecular interactions.

Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry.

Protein surfaces are important in most biological processes, including protein-protein interactions, enzymatic catalysis, and protein-ligand binding. We report a method in which hydroxyl radicals generated by a rapid-UV irradiation of a 15% hydrogen peroxide solution were utilized to oxidize specific amino acid side chains of two model proteins (lysozyme, beta-lactoglobulin A), according to the residues' chemical reactivities and the solvent accessibility of the reactive carbons and sulfurs in the residue. Oxidized peptides generated by tryptic digestion were identified by electrospray-Fourier transform mass spectrometry.

Identification of phosphorylation sites on the yeast ribonucleotide reductase inhibitor Sml1.

Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway.

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.

Protein surface mapping by chemical oxidation: structural analysis by mass spectrometry.

The solvent-accessible surface area of proteins is important in biological function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chemical technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidation. Under conditions of low protein oxidation (zero to three oxygen atoms added per apomyoglobin molecule), we have positively identified five oxidation sites by liquid chromatography-tandem mass spectrometry and high-resolution Fourier transform mass spectrometry.

Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase.

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products.