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1.  Controlled Measurement and Comparative Analysis of Cellular Components in E. coli Reveals Broad Regulatory Changes in Response to Glucose Starvation 
PLoS Computational Biology  2015;11(8):e1004400.
How do bacteria regulate their cellular physiology in response to starvation? Here, we present a detailed characterization of Escherichia coli growth and starvation over a time-course lasting two weeks. We have measured multiple cellular components, including RNA and proteins at deep genomic coverage, as well as lipid modifications and flux through central metabolism. Our study focuses on the physiological response of E. coli in stationary phase as a result of being starved for glucose, not on the genetic adaptation of E. coli to utilize alternative nutrients. In our analysis, we have taken advantage of the temporal correlations within and among RNA and protein abundances to identify systematic trends in gene regulation. Specifically, we have developed a general computational strategy for classifying expression-profile time courses into distinct categories in an unbiased manner. We have also developed, from dynamic models of gene expression, a framework to characterize protein degradation patterns based on the observed temporal relationships between mRNA and protein abundances. By comparing and contrasting our transcriptomic and proteomic data, we have identified several broad physiological trends in the E. coli starvation response. Strikingly, mRNAs are widely down-regulated in response to glucose starvation, presumably as a strategy for reducing new protein synthesis. By contrast, protein abundances display more varied responses. The abundances of many proteins involved in energy-intensive processes mirror the corresponding mRNA profiles while proteins involved in nutrient metabolism remain abundant even though their corresponding mRNAs are down-regulated.
Author Summary
Bacteria frequently experience starvation conditions in their natural environments. Yet how they modify their physiology in response to these conditions remains poorly understood. Here, we performed a detailed, two-week starvation experiment in E. coli. We exhaustively monitored changes in cellular components, such as RNA and protein abundances, over time. We subsequently compared and contrasted these measurements using novel computational approaches we developed specifically for analyzing gene-expression time-course data. Using these approaches, we could identify systematic trends in the E. coli starvation response. In particular, we found that cells systematically limit mRNA and protein production, degrade proteins involved in energy-intensive processes, and maintain or increase the amount of proteins involved in energy production. Thus, the bacteria assume a cellular state in which their ongoing energy use is limited while they are poised to take advantage of any nutrients that may become available.
PMCID: PMC4537216  PMID: 26275208
2.  A Theoretical Justification for Single Molecule Peptide Sequencing 
PLoS Computational Biology  2015;11(2):e1004080.
The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences) to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.
Author Summary
The development of next-generation DNA and RNA sequencing methods has transformed biology, with current platforms generating >1 billion sequencing reads per run. Unfortunately, no method of similar scale and throughput exists to identify and quantify specific proteins in complex mixtures, representing a critical bottleneck in many biochemical and molecular diagnostic assays. What is urgently needed is a massively parallel method, akin to next-gen DNA sequencing, for identifying and quantifying peptides or proteins in a sample. In principle, single-molecule peptide sequencing could achieve this goal, allowing billions of distinct peptides to be sequenced in parallel and thereby identifying proteins composing the sample and digitally quantifying them by direct counting of peptides. Here, we discuss theoretical considerations of single molecule peptide sequencing, suggest one possible experimental strategy, and, using computer simulations, characterize the potential utility and unusual properties of this future proteomics technology.
PMCID: PMC4341059  PMID: 25714988
3.  Age-Dependent Evolution of the Yeast Protein Interaction Network Suggests a Limited Role of Gene Duplication and Divergence 
PLoS Computational Biology  2008;4(11):e1000232.
Proteins interact in complex protein–protein interaction (PPI) networks whose topological properties—such as scale-free topology, hierarchical modularity, and dissortativity—have suggested models of network evolution. Currently preferred models invoke preferential attachment or gene duplication and divergence to produce networks whose topology matches that observed for real PPIs, thus supporting these as likely models for network evolution. Here, we show that the interaction density and homodimeric frequency are highly protein age–dependent in real PPI networks in a manner which does not agree with these canonical models. In light of these results, we propose an alternative stochastic model, which adds each protein sequentially to a growing network in a manner analogous to protein crystal growth (CG) in solution. The key ideas are (1) interaction probability increases with availability of unoccupied interaction surface, thus following an anti-preferential attachment rule, (2) as a network grows, highly connected sub-networks emerge into protein modules or complexes, and (3) once a new protein is committed to a module, further connections tend to be localized within that module. The CG model produces PPI networks consistent in both topology and age distributions with real PPI networks and is well supported by the spatial arrangement of protein complexes of known 3-D structure, suggesting a plausible physical mechanism for network evolution.
Author Summary
Proteins function together forming stable protein complexes or transient interactions in various cellular processes, such as gene regulation and signaling. Here, we address the basic question of how these networks of interacting proteins evolve. This is an important problem, as the structures of such networks underlie important features of biological systems, such as functional modularity, error-tolerance, and stability. It is not yet known how these network architectures originate or what driving forces underlie the observed network structure. Several models have been proposed over the past decade—in particular, a “rich get richer” model (preferential attachment) and a model based upon gene duplication and divergence—often based only on network topologies. Here, we show that real yeast protein interaction networks show a unique age distribution among interacting proteins, which rules out these canonical models. In light of these results, we developed a simple, alternative model based on well-established physical principles, analogous to the process of growing protein crystals in solution. The model better explains many features of real PPI networks, including the network topologies, their characteristic age distributions, and the spatial distribution of subunits of differing ages within protein complexes, suggesting a plausible physical mechanism of network evolution.
PMCID: PMC2583957  PMID: 19043579

Results 1-3 (3)