Mary S. Lipton

An accurate mass tag strategy for quantitative and high-throughput proteome measurements

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide “accurate mass tags” (AMTs) produced by global protein enzymatic digestion. The two‐stage strategy exploits Fourier transform‐ion cyclotron resonance (FT‐ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from “potential mass tags” identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT‐ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 105 components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.

Fermentation, Hydrogen, and Sulfur Metabolism in Multiple Uncultivated Bacterial Phyla

Bacterial PERegrinations Many branches of the bacterial domain of life are only known from sequences that turn up in metagenomic analyses and are still only named by acronym—for example, the phylum-level groups BD1-5, OP11, OD1, and the PERs. The parent organisms are probably widespread, but they have not been cultured, and very little is known about their metabolisms or their contributions and functions in the natural environment. Wrighton et al. (p. 1661) pumped acetate into an aquifer in Colorado to prompt the naturally occurring bacteria into action and then, from the runoff, filtered out the smaller microbial cells for further analysis. Mass-spectrometry–based proteomics was used to test for functional activity, and 49 distinct genomes were recovered, many with surprising functional attributes. All of the recovered organisms appeared to be strict anaerobes with a full glycolytic pathway that were capable of augmenting energy production by coupling proton-pumping activity to adenosine triphosphate synthase. Several hydrogenases were found that seemed to be able to switch between hydrogen production and polysulfide reduction, depending on the substrate available. Notably, carbon dioxide assimilation was a common feature, with many genes having similarity to those of archaea. Near-complete reconstruction of the genomes of 21 widespread uncultured environmental bacteria reveals metabolic novelties. BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like hybrid type II/III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO2 fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.

Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Transport functions dominate the SAR11 metaproteome at low-nutrient extremes in the Sargasso Sea

The northwestern Sargasso Sea undergoes annual cycles of productivity with increased production in spring corresponding to periods of upwelling, and oligotrophy in summer and autumn, when the water column becomes highly stratified. The biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorus and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary liquid chromatography (LC)-tandem mass spectrometry to detect microbial proteins in surface samples collected in September 2005. A total of 2215 peptides that mapped to 236 SAR11 proteins, 1911 peptides that mapped to 402 Prochlorococcus proteins and 2407 peptides that mapped to 404 Synechococcus proteins were detected. Mass spectra from SAR11 periplasmic substrate-binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars and spermidine. Proteins implicated in the prevention of oxidative damage and protein refolding were also abundant. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme, but nutrient flux is sufficient to sustain microbial community activity.

Global Analysis of Deinococcus Radiodurans Proteome by Csing Accurate Mass Tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms: Characterization by Infrared Spectroscopy and Proteomics

The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR-1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal. Methods and Principal Findings We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject. Conclusions Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.

Proteogenomics: needs and roles to be filled by proteomics in genome annotation

While genome sequencing efforts reveal the basic building blocks of life, a genome sequence alone is insufficient for elucidating biological function. Genome annotation--the process of identifying genes and assigning function to each gene in a genome sequence--provides the means to elucidate biological function from sequence. Current state-of-the-art high-throughput genome annotation uses a combination of comparative (sequence similarity data) and non-comparative (ab initio gene prediction algorithms) methods to identify protein-coding genes in genome sequences. Because approaches used to validate the presence of predicted protein-coding genes are typically based on expressed RNA sequences, they cannot independently and unequivocally determine whether a predicted protein-coding gene is translated into a protein. With the ability to directly measure peptides arising from expressed proteins, high-throughput liquid chromatography-tandem mass spectrometry-based proteomics approaches can be used to verify coding regions of a genomic sequence. Here, we highlight several ways in which high-throughput tandem mass spectrometry-based proteomics can improve the quality of genome annotations and suggest that it could be efficiently applied during the gene calling process so that the improvements are propagated through the subsequent functional annotation process.

Proteogenomic Monitoring of Geobacter Physiology during Stimulated Uranium Bioremediation

ABSTRACT Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant Geobacter community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic Geobacter-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate Geobacter genomes. Genome-specific peptides indicate early proliferation of multiple M21 and Geobacter bemidjiensis-like strains and later possible emergence of M21 and G. bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving Geobacter strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.

Mass spectrometic detection for capillary isoelectric focusing separations of complex protein mixtures

Capillary isoelectric focusing (CIEF) can provide high‐resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information‐rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400—1000 putative proteins in the mass range of 2—100 kDa from total injections of ˜ 300 ng protein in single CIEF‐FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome‐wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.