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Jeffrey C Smith

Publications and source records attributed to Jeffrey C Smith.

10 recordsLinked to original sources
PubMed2007

Multiplexed proteomic reactor for the processing of proteomic samples.

We report the development of a 96-well plate proteomic reactor for gel-free processing of minute amounts of complex proteomic samples. The device performs multiplexed trapping, enrichment, and biochemical processing of proteins, resulting in concentrated peptide solutions ready for mass spectrometric analysis. Individual wells on the reactor can process up to 2 microg of protein. We also report the coupling of the plate proteomic reactor with protein fractionation using size-exclusion chromatography for large-scale identification of proteins. To illustrate the potential of this approach, we separated 400 microg of MCF7 cell lysate using size-exclusion chromatography and processed 35 protein fractions on the reactor plate. Using stringent criteria when searching the data, a total of 875 unique proteins were identified. More relaxed searching conditions associated with a 1% false positive rate led to the identification of 2683 unique proteins, meaning that one protein was identified per 3-10 ng of total protein lysate loaded on the reactor plate.

Biomarkers, Tumor↗
PubMed2007

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells.

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

Adenosine Triphosphatases↗
PubMed2006

Proteomics technology in systems biology.

It has now become apparent that a full understanding of a biological process (e.g. a disease state) is only possible if all biomolecular interactions are taken into account. Systems biology works towards understanding the intricacies of cellular life through the collaborative efforts of biologists, chemists, mathematicians and computer scientists and recently, a number of laboratories around the world have embarked upon such research agendas. The fields of genomics and proteomics are foundational in systems biology studies and a great deal of research is currently being conducted in each worldwide. Moreover, many technological advances (particularly in mass spectrometry) have led to a dramatic rise in the number of proteomic studies over the past two decades. This short review summarizes a selection of technological innovations in proteomics that contribute to systems biology studies.

Chromatography, Affinity↗
PubMed2006

Respiratory rhythm generation during gasping depends on persistent sodium current.

In severe hypoxia, homeostatic mechanisms maintain function of the brainstem respiratory network. We hypothesized that hypoxia involves a transition from neuronal mechanisms of normal breathing (eupnea) to a rudimentary pattern of inspiratory movements (gasping). We provide evidence for hypoxia-driven transformation within the central respiratory oscillator, in which gasping relies on persistent sodium current, whereas eupnea does not depend on this cellular mechanism.

Action Potentials↗
PubMed2004

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: dimerization, complexation, and dissociation.

The investigation of protein quaternary structure, protein-cofactor, and protein-ligand interactions by mass spectrometry is often limited by the fragility of such interactions under experimental conditions. To develop more gentle conditions of perhaps general use, we used as a model for study the oxygenase domain of murine inducible nitric oxide synthase (iNOS), which is homodimeric, binds heme and tetrahydrobiopterin H(4)B cofactors, and the substrate L-arginine. The energetics of the collisions in q2 and in the lens region of the mass spectrometer were manipulated for varying the degree of solvation around the non-covalently bound ions. Furthermore, the number of low-energy collisions in the collision cell of the instrument was varied, focusing and dampening the ion beam. Under gentle source collision conditions, and using multiple low-energy collisions in the collision cell of the mass spectrometer, dimers of the iNOS oxygenase domain containing heme, H(4)B, and arginine were observed intact after electrospraying at pH values near neutrality; a mutant of this protein (Trp188 --> Phe) was monomeric and did not bind cofactors. The pH dependence of the iNOS oxygenase domain under acidic conditions was also studied; while heme remained bound to the protein between pH 2.5 and 4.0, the dimeric structure was disrupted. Our findings confirm that non-covalently bound macromolecular complexes are retained and observable using electrospray mass spectrometry under the appropriate experimental conditions.

Cold Temperature↗
PubMed2002

Periodicity, mixed-mode oscillations, and quasiperiodicity in a rhythm-generating neural network.

We studied patterns of oscillatory neural activity in the network that generates respiratory rhythm in mammals. When isolated in vitro, this network spontaneously generates an inspiratory-related motor rhythm, with stable amplitude from cycle to cycle. We show that progressively elevating neuronal excitability in vitro causes periodic modulation of this inspiratory rhythm, evoking (in order): mixed-mode oscillations, quasiperiodicity, and ultimately disorganized aperiodic activity. Thus, the respiratory network oscillator follows a well defined sequence of behavioral states characterized by dynamical systems theory, which includes discrete stages of periodic and quasiperiodic amplitude modulation and progresses (according to theory) to aperiodic chaos-like behavior. We also observed periodic, mixed-mode periodic, and quasiperiodic breathing patterns in neonatal rodents and human infants in vivo, suggesting that breathing patterns generated by the intact nervous system reflect deterministic neural activity patterns in the underlying rhythm-generating network.

Animals↗
PubMed2002

Persistent sodium current, membrane properties and bursting behavior of pre-bötzinger complex inspiratory neurons in vitro.

We measured persistent Na(+) current and membrane properties of bursting-pacemaker and nonbursting inspiratory neurons of the neonatal rat pre-Bötzinger complex (pre-BötC) in brain stem slice preparations with a rhythmically active respiratory network in vitro. In whole-cell recordings, slow voltage ramps (</=100 mV/s) inactivated the fast, spike-generating Na(+) current and yielded N-shaped current-voltage relationships with nonmonotonic, negative-slope regions between -60 and -35 mV when the voltage-sensitive component was isolated. The underlying current was a TTX-sensitive persistent Na(+) current (I(NaP)) since the inward current was present at slow voltage ramp speeds (3.3-100 mV/s) and the current was blocked by 1 microM TTX. We measured the biophysical properties of I(NaP) after subtracting the voltage-insensitive "leak" current (I(Leak)) in the presence of Cd(2+) and in some cases tetraethylammonium (TEA). Peak I(NaP) ranged from -50 to -200 pA at a membrane potential of -30 mV. Decreasing the speed of the voltage ramp caused time-dependent I(NaP) inactivation, but this current was present at ramp speeds as low as 3.3 mV/s. I(NaP) activated at -60 mV and obtained half-maximal activation near -40 mV. The subthreshold voltage dependence and slow inactivation kinetics of I(NaP), which closely resemble those of I(NaP) mathematically modeled as a burst-generation mechanism in pacemaker neurons of the pre-BötC, suggest that I(NaP) predominantly influences bursting dynamics of pre-BötC inspiratory pacemaker neurons in vitro. We also found that the ratio of persistent Na(+) conductance to leak conductance (g(NaP)/g(Leak)) can distinguish the phenotypic subpopulations of bursting pacemaker and nonbursting inspiratory neurons: pacemaker neurons showed g(NaP)/g(Leak) > g(NaP)/g(Leak) in nonpacemaker cells (P < 0.0002). We conclude that I(NaP) is ubiquitously expressed by pre-BötC inspiratory neurons and that bursting pacemaker behavior within the heterogeneous population of inspiratory neurons is achieved with specific ratios of these two conductances, g(NaP) and g(Leak).

Animals↗
PubMedDate not supplied

Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila.

To improve the utility of increasingly large numbers of available unannotated and initially poorly annotated genomic sequences for proteome analysis, we demonstrate that effective protein identification can be made on a large and unannotated genome. The strategy developed is to translate the unannotated genome sequence into amino acid sequence encoding putative proteins in all six reading frames, to identify peptides by tandem mass spectrometry (MS/MS), to localize them on the genome sequence, and to preliminarily annotate the protein via a similarity search by BLAST. These tasks have been optimized and automated. Optimization to obtain multiple peptide matches in effect extends the searchable region and results in more robust protein identification. The viability of this strategy is demonstrated with the identification of 223 cilia proteins in the unicellular eukaryotic model organism Tetrahymena thermophila, whose initial genomic sequence draft was released in November 2003. To the best of our knowledge, this is the first demonstration of large-scale protein identification based on such a large, unannotated genome. Of the 223 cilia proteins, 84 have no similarity to proteins in NCBI's nonredundant (nr) database. This methodology allows identifying the locations of the genes encoding these novel proteins, which is a necessary first step to downstream functional genomic experimentation.

Amino Acid Sequence↗
PubMedDate not supplied

Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry.

Post-translational modification of proteins via the covalent attachment of Ubiquitin (Ub) plays an important role in the regulation of protein stability and function in eukaryotic cells. In the present study, we describe a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We have demonstrated the applicability of this approach by identifying 70 ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. This method will aid the study of protein ubiquitination and may be used as a tool for the discovery of novel biomarkers that are associated with disease progression.

Biomarkers, Tumor↗