Daniel Lopez-Ferrer

Omic data from evolved E. coli are consistent with computed optimal growth from genome‐scale models

After hundreds of generations of adaptive evolution at exponential growth, Escherichia coli grows as predicted using flux balance analysis (FBA) on genome‐scale metabolic models (GEMs). However, it is not known whether the predicted pathway usage in FBA solutions is consistent with gene and protein expression in the wild‐type and evolved strains. Here, we report that >98% of active reactions from FBA optimal growth solutions are supported by transcriptomic and proteomic data. Moreover, when E. coli adapts to growth rate selective pressure, the evolved strains upregulate genes within the optimal growth predictions, and downregulate genes outside of the optimal growth solutions. In addition, bottlenecks from dosage limitations of computationally predicted essential genes are overcome in the evolved strains. We also identify regulatory processes that may contribute to the development of the optimal growth phenotype in the evolved strains, such as the downregulation of known regulons and stringent response suppression. Thus, differential gene and protein expression from wild‐type and adaptively evolved strains supports observed growth phenotype changes, and is consistent with GEM‐computed optimal growth states.

Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells†

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed‐phase liquid chromatography strategy as a first dimension for two‐dimensional liquid chromatography tandem mass spectrometry (“shotgun”) proteomic analysis of trypsin‐digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed‐phase liquid chromatography as a first‐dimension fractionation strategy resulted in 1.8‐ and 1.6‐fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed‐phased strategy is an attractive alternative to strong cation exchange for two‐dimensional shotgun proteomic analysis.

Improved Method for Differential Expression Proteomics Using Trypsin-catalyzed 18O Labeling with a Correction for Labeling Efficiency

Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 μg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.

Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion

A stable and robust trypsin‐based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300‐fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC‐MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in “real‐world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Properties of Average Score Distributions of SEQUEST The Probability Ratio Method

High throughput identification of peptides in databases from tandem mass spectrometry data is a key technique in modern proteomics. Common approaches to interpret large scale peptide identification results are based on the statistical analysis of average score distributions, which are constructed from the set of best scores produced by large collections of MS/MS spectra by using searching engines such as SEQUEST. Other approaches calculate individual peptide identification probabilities on the basis of theoretical models or from single-spectrum score distributions constructed by the set of scores produced by each MS/MS spectrum. In this work, we study the mathematical properties of average SEQUEST score distributions by introducing the concept of spectrum quality and expressing these average distributions as compositions of single-spectrum distributions. We predict and demonstrate in the practice that average score distributions are dominated by the quality distribution in the spectra collection, except in the low probability region, where it is possible to predict the dependence of average probability on database size. Our analysis leads to a novel indicator, the probability ratio, which takes optimally into account the statistical information provided by the first and second best scores. The probability ratio is a non-parametric and robust indicator that makes spectra classification according to parameters such as charge state unnecessary and allows a peptide identification performance, on the basis of false discovery rates, that is better than that obtained by other empirical statistical approaches. The probability ratio also compares favorably with statistical probability indicators obtained by the construction of single-spectrum SEQUEST score distributions. These results make the robustness, conceptual simplicity, and ease of automation of the probability ratio algorithm a very attractive alternative to determine peptide identification confidences and error rates in high throughput experiments.

Nanobiocatalysis for protein digestion in proteomic analysis

The process of protein digestion is a critical step for successful protein identification in bottom‐up proteomic analyses. To substitute the present practice of in‐solution protein digestion, which is long, tedious, and difficult to automate, many efforts have been dedicated for the development of a rapid, recyclable and automated digestion system. Recent advances of nanobiocatalytic approaches have improved the performance of protein digestion by using various nanomaterials such as nanoporous materials, magnetic nanoparticles, and polymer nanofibers. Especially, the unprecedented success of trypsin stabilization in the form of trypsin‐coated nanofibers, showing no activity decrease under repeated uses for 1 year and retaining good resistance to proteolysis, has demonstrated its great potential to be employed in the development of automated, high‐throughput, and on‐line digestion systems. This review discusses recent developments of nanobiocatalytic approaches for the improved performance of protein digestion in speed, detection sensitivity, recyclability, and trypsin stability. In addition, we also introduce approaches for protein digestion under unconventional energy input for protein denaturation and the development of microfluidic enzyme reactors that can benefit from recent successes of these nanobiocatalytic approaches.

Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestion and selected MS/MS ion monitoring.

A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only 11 peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers.

On-line digestion system for protein characterization and proteome analysis

An efficient on-line digestion system that reduces the number of sample manipulation steps has been demonstrated for high-throughput proteomics. By incorporating a pressurized sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, yet rapid digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated online pressurized digestion system coupled to an ion mobility time-of-flight mass spectrometer, an ion trap mass spectrometer, or both. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to on-line high-throughput applications. In addition to simplifying and expediting sample processing, the system was easy to implement and no cross-contamination was observed among samples. As a result, the online digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research (rapid screening of protein-based drugs).

Pressurized Pepsin Digestion in Proteomics AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.

Aging Enhances the Production of Reactive Oxygen Species and Bactericidal Activity in Peritoneal Macrophages by Upregulating Classical Activation Pathways

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.