Biomedical subjects

Kelvin H Lee

Publications and source records attributed to Kelvin H Lee.

At least 19 recordsLinked to original sources

PubMed2007

Cerebrospinal fluid proteomic biomarkers for Alzheimer's disease.

OBJECTIVE: To find a panel of proteins in antemortem cerebrospinal fluid (CSF) that could be used to differentiate between samples from Alzheimer's disease (AD) patients and samples from healthy and neurological control subjects. METHODS: For a test cohort, antemortem CSF proteins from 34 AD and 34 non-AD patients were separated using two-dimensional gel electrophoresis. The resulting protein patterns were analyzed using the random forest multivariate statistical method. Protein spots of interest were identified using tandem time-of-flight mass spectrometry. A validation cohort consisting of CSF from 18 AD patients and 10 non-AD subjects was analyzed in a similar way. RESULTS: Using the test cohort, a panel of 23 spots was identified that could be used to differentiate AD and non-AD gels with a sensitivity of 94%, a specificity of 94%, and a predicted classification error rate of only 5.9%. These proteins are related to the transport of beta-amyloid, the inflammatory response, proteolytic inhibition, and neuronal membrane proteins. The 23 spots separately classified the validation cohort with 90% sensitivity (probable AD subjects), 83% specificity, and a predicted classification error rate of 14% in a blinded analysis. The total observed sensitivity is 93%, the total observed specificity is 90%, and the predicted classification error rate is 8.3%. INTERPRETATION: A panel of possible CSF biomarkers for AD has been identified using proteomic methods.

Alzheimer Disease↗

PubMed2006

Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1.

The proteomic response of recombinant Escherichia coli producing human glucagon-like peptide-1 was analyzed by two-dimensional gel electrophoresis. Protein spots in two-dimensional gel could be identified by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their expression profiles were compared with those of nonproducing cells. Thirty-five intracellular proteins exhibited differential expression levels between the production and control strains. These changes reflected physiological responses to heterologous peptide production in recombinant E. coli. Specifically, physiological changes included the down-regulation of proteins involved in the central carbon metabolism, biosynthesis of cellular building blocks and peptides, and up-regulation of cell protection proteins and some sugar transport proteins. This comprehensive analysis would provide useful information for understanding physiological alterations to heterologous peptide production and for designing efficient metabolic engineering strategies for the production of recombinant peptides in E. coli.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Proteomic analysis of cerebrospinal fluid changes related to postmortem interval.

BACKGROUND: The study of proteins with altered production in postmortem cerebrospinal fluid (CSF) compared with antemortem CSF may improve the understanding of biochemical changes that occur immediately after death. METHODS: Two CSF samples (1 antemortem and 1 postmortem) were collected from 7 patients and analyzed by 2-dimensional gel electrophoresis. An analysis was also performed to identify proteins that showed a correlation between concentration change and postmortem interval. Tandem mass spectrometry was used to identify the proteins. RESULTS: Fifty-four protein spots were identified that showed a consistent and significant change in concentration in the postmortem CSF of all 7 patients (>3.5-fold, P <0.01). The proteins in these spots derive from a variety of functional groups, including cytoskeletal proteins, enzymes involved in glycolysis, and proteins that prevent oxidative stress. Fourteen protein spots were found to have an increase in production that correlated with postmortem interval. CONCLUSIONS: Changes in protein production of postmortem vs antemortem CSF were studied. The proteins observed to change production in the postmortem CSF include several proteins previously observed as potential stroke biomarkers.

Aged↗

PubMed2006

Shotgun proteomics using the iTRAQ isobaric tags.

Shotgun proteomic methods involving isobaric tagging of peptides enable high-throughput proteomic analysis. iTRAQ reagents allow simultaneous identification and quantitation of proteins in four different samples using tandem mass spectrometry (MS). In this article, we provide a brief description of proteome analysis using iTRAQ reagents and review the current applications of these reagents in proteomic studies. We also compare different aspects of protein identification including protein sequence coverage and proteome coverage obtained using iTRAQ reagents with those using other shotgun proteomic techniques. We briefly discuss the issue of isotope purity correction in measured peak areas during protein quantitation using iTRAQ reagents. Finally, we conclude with some of the current challenges in MS-based proteomic analysis that are limiting protein identifications obtained by different shotgun proteomic methods.

Animals↗

PubMed2006

Two-dimensional protein electrophoresis: from molecular pathway discovery to biomarker discovery in neurological disorders.

Two-dimensional protein electrophoresis (2-DE) has undergone many technical improvements in the past 30 years, resulting in an analytical method that is unparalleled in the resolution of complex protein mixtures and capable of quantifying changes in protein expression from a wide variety of tissues and samples. The technique has been applied in many studies of neurologic disease to identify changes in spot patterns that correlate with disease. The true power of the technique emerges when it is coupled to state-of-the-art methods in mass spectrometry, which enable identification of the protein or proteins contained within a spot of interest on a 2-DE map. Investigators have successfully applied the technique to gain improved understanding of neurologic disease mechanisms in humans and in animal models and to discover biomarkers that are useful in the clinical setting. An important extension to these efforts that has not been realized thus far is the desire to profile changes in protein expression that result from therapy to help relate disease-modifying effects at the molecular level with clinical outcomes. Here we review the major advances in 2-DE methods and discuss specific examples of its application in the study of neurologic diseases.

Animals↗

PubMed2005

A surface modification strategy on silicon nitride for developing biosensors.

A surface modification strategy for the use of giant magnetoresistive materials in the detection of protein-protein interactions is developed. This modification strategy is based on silanization of semiconductive materials. A native silicon nitride surface was treated with concentrated hydrofluoric acid to improve surface homogeneity. Nano-strip was used to oxidize silicon nitride to form a hydrophilic layer. Aminopropyltriethoxysilane was subsequently used to functionalize the treated surfaces to form amine groups, which were further activated with glutaraldehyde to introduce a layer of aldehyde groups. The effectiveness of this modification strategy was validated by chemiluminescence immunoassays of purified 6x His-HrpW of Pseudomonas syringae pv. tomato DC3000 and human transferrin. Signals with intensities related to concentrations of these two immobilized model proteins were observed. The modified surface was also validated by a more complex system: intercellular proteins secreted by DC3000. HrpW in these protein mixtures was successfully recognized by anti-HrpW antibodies when mixed proteins were immobilized onto activated surfaces. This surface modification strategy provides a platform onto which proteins can be directly immobilized for biosensor and protein array applications.

Biosensing Techniques↗

PubMed2005

A polymeric microchip with integrated tips and in situ polymerized monolith for electrospray mass spectrometry.

We describe the integration of a cyclo-olefin polymer based microchip with a sheathless capillary tip for electrospray ionization-mass spectrometry (ESI-MS). The microchip was fabricated by hot embossing and thermal bonding. Its design includes a side channel for adjusting the composition of the electrospray solution so that analytes in 100% water can be analyzed. The fused silica capillaries, used for sample introduction, and the electrospray tips for MS coupling were directly inserted into the microchannel before thermal bonding of the device. A microfabricated on-chip gold microelectrode was used to apply the electrospray voltage. Annealing the device after thermal bonding increased the pressure resistance of the microchip. The cross section of the microchannel was imaged by scanning electron microscopy to estimate the effects of the annealing step. The relationship between the applied electrospray voltages and MS signal was measured at different flow rates by coupling the device to an ion trap mass spectrometer. The performance of the microchip was evaluated by MS analysis of imipramine in ammonium acetate buffer solution by direct infusion. An alkylacrylate based monolith polymer bed for on-chip sample pretreatment and separation was polymerized in the microchannel and tested for ESI-MS applications.

Journal Article↗

PubMed2005

Cytochalasin D can improve heterologous protein productivity in adherent Chinese hamster ovary cells.

We generated a series of adherent gene-amplified CHO clones expressing human secreted alkaline phosphatase (SEAP) as a model for heterologous protein production. Clones demonstrate a 26- to 52-fold increase in productivity compared to controls after dhfr/methotrexate-mediated gene amplification and clone selection. SEAP is stably expressed in these clones over at least a 6-week period without significant productivity loss. Two-dimensional protein electrophoresis identified 21 proteins that exhibited altered expression in clones of increasing SEAP productivity. Based on MALDI TOF/TOF mass spectrometry of relevant protein spots, changes in translation, energy pathways, chaperones, regulatory proteins, and cytoskeletal proteins were observed, including a 4-fold expression increase in actin capping protein. We hypothesized that an alteration of the actin cytoskeleton using cytochalasin D as a mimic for actin-capping protein could have a beneficial effect on heterologous protein secretion. Treatment with 0.5 mug/mL cytochalasin D increased SEAP productivity 2- to 3-fold compared to an amplified control which resulted in an increase in productivity from 52- to 150-fold compared to a nonamplified parent.

Animals↗

PubMed2005

Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses.

Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification. However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression. The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community.

Computer Simulation↗

PubMed2005

Isoelectric focusing in cyclic olefin copolymer microfluidic channels coated by polyacrylamide using a UV photografting method.

As an alternative material to glass or silicon, microfluidic devices made from a cyclic olefin copolymer (COC) were fabricated. This material is of interest because of the relative ease of fabrication, low costs, and solvent resistance. However, as a result of the strong hydrophobic interactions normally present, COC surfaces are not suitable for protein separations. To reduce the protein adsorption and make COC suitable for protein separations, UV-initiated grafting of polyacrylamide was used to coat the surface of COC devices. The change in surface properties caused by different graft times was studied. The surface hydrophilicity and electroosmotic mobility were characterized by contact angle and electroosmosis measurements. Isoelectric focusing was performed to test protein separations in polyacrylamide-coated COC microchannels. A single protein, carbonic anhydrase, was used to analyze the focusing effects and peak capacities in uncoated and polyacrylamide-coated COC devices. Peak capacities ranging from 75 to 190 were achieved with a polyacrylamide-coated surface. A mixture of two proteins, conalbumin labeled with Alexa Fluor 488 and beta-lactoglobulin A labeled with Alexa Fluor 546, was used to test protein separations. Linear and rapid separation of proteins was achieved in the polyacrylamide-coated COC microfluidic device.

Acrylic Resins↗

PubMed2005

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells.

An important consideration in the measurement of quantitative changes in protein expression is the consistency of the observations for a given technique as well as the reproducibility of the experiment. A quantitative assessment of the technical and biological variability is crucial to avoid erroneous inferences and conclusions. Two methods for measuring quantitative changes in protein expression are two-dimensional electrophoresis (2-DE) and shotgun proteomics of isobaric-tagged samples using iTRAQ reagents. An assessment of changes in Escherichia coli protein expression in response to rhsA induction demonstrates that half of the quantified protein expression ratios have a coefficent of variation (CV) less than 0.31 using 2-DE and less than 0.24 using isobaric tags; whereas 95% of the quantified protein expression ratios have a CV less than 0.81 using 2-DE and less than 0.53 using isobaric tags. The selective removal of outlier data points from the shotgun method using Grubb's and Rosner's statistical outlier tests improves the consistency of the quantitation data obtained.

Chromatography, Liquid↗

PubMed2005

Coupling on-chip solid-phase extraction to electrospray mass spectrometry through an integrated electrospray tip.

We report the integration of solid-phase extraction (SPE) with mass spectrometry (MS) through an on-chip electrospray tip for sample precleaning and preconcentration. An in situ polymerized alkylacrylate-based monolithic column was used as the stationary phase for the on-chip SPE. Each microchip consists of two sets of microchannels and their respective integrated electrospray tips, with a common gold electrode. After the microchip was fabricated from cycloolefin polymer by hot embossing, thermal bonding, and annealing steps, a mixture of monomers and porogenic solvents was pumped into the microchannels and certain areas of the main microchannels were exposed to UV irradiation through a mask. The resulting porous monolithic beds that were polymerized from different compositions of the mixture were characterized by scanning electron microscopy. The microchip containing the monolithic column was then interfaced to an ion trap (IT) mass spectrometer by modifying a commercially available interfacing system. Makeup solution from the side channel was infused concurrently with the solution flowing into the main channel, and the mixture of these two solutions was sprayed into the MS orifice. Both the adsorption and elution of a pharmaceutical test compound, imipramine, to and from the on-chip SPE columns were monitored by MS. The potential application of this device for sample cleanup was demonstrated by pretreatment of urine samples spiked with imipramine.

Humans↗

PubMed2005

Quantitative analysis of protein expression using amine-specific isobaric tags in Escherichia coli cells expressing rhsA elements.

We describe the use of amine-specific isobaric tags for protein expression quantification to study the effect of rhsA element over-expression in Escherichia coli. The use of an isobaric tagging strategy facilitates a shotgun approach to proteomic analysis and enables quantitation of up to four samples in parallel, based on the reporter ion series using tandem mass spectrometry (MS/MS). Using a liquid chromatography matrix-assisted laser desorption/ionization approach, 23,139 MS/MS spectra were collected. Five thousand sixty-three peptides derived from 780 proteins were quantified including several lower abundance proteins, such as transcription factors, DnaB and DnaG. More than 65% of the proteins had at least two high confidence peptide matches per protein (p<0.05). Further, a statistical test based on the Grubb's and Rosner's tests was able to discriminate outlier data. The removal of outlier data had no significant effect on the functional categories of proteins that were represented in the study.

Amines↗

PubMed2005

Kinetic characterization of sequencing grade modified trypsin.

Prior to analysis by mass spectrometry, protein samples are often digested. Maximizing the peptide yield from digestion can increase the number of peptides detected and the confidence in protein identification. To determine the optimal conditions for digestion, the Michaelis-Menten kinetic parameters for Promega sequencing grade modified trypsin were measured over a range of temperatures and pHs. The results indicate that an increase in digestion temperature above 37 degrees C, the temperature traditionally used in digestion methods, could offer an increase in peptides detected.

Caseins↗

PubMed2005

Complement protein isoforms in CSF as possible biomarkers for neurodegenerative disease.

It has been suggested that the activation of the complement system is involved in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Here, the CSF expression levels of complement proteins C3b, C4b, factor B, and factor H were compared between normal subjects and patients diagnosed with AD, PD, MS, and neurosyphilis. The CSF proteins were initially separated using two-dimensional gel electrophoresis, which allowed the comparison of some of the individual complement isoforms. Patients with AD, PD, and MS all showed more than one complement isoform with a significant change (p < 0.05) in CSF expression level compared to normal subjects. PD patients were found to have the greatest number of significantly changed isoforms, all showing a decreased expression level in PD CSF. The complement isoforms examined were able to distinguish between some, but not all, of the diseases studied. The data suggest that when investigating a protein as a possible biomarker, it may be useful to compare individual protein isoform expression levels in addition to the more commonly measured total protein expression level.

Alzheimer Disease↗

PubMed2005

Improved understanding of gene expression regulation using systems biology.

This article reviews the current state of systems biology approaches, including the experimental tools used to generate 'omic' data and computational frameworks to interpret this data. Through illustrative examples, systems biology approaches to understand gene expression and gene expression regulation are discussed. Some of the challenges facing this field and the future opportunities in the systems biology era are highlighted.

Animals↗

PubMed2005

Alzheimer's disease cerebrospinal fluid biomarker discovery: a proteomics approach.

There is significant interest in the identification of effective biomarkers for Alzheimer's disease. Such biomarkers could aid in the clinical diagnosis of the disease and may be useful in assessing the efficacy of various treatment strategies. The search for biomarkers often includes the analysis of changes in cerebrospinal fluid protein expression that correlate with disease. These changes can be measured using a variety of technologies for protein expression profiling. Although there is great promise in the application of these methods to biomarker discovery based on some preliminary observations, there are significant issues in the capabilities of most of these technologies that have limited their effective application. The most recent literature involving proteomic discovery of new cerebrospinal fluid biomarkers for Alzheimer's disease is reviewed.

Alzheimer Disease↗

PubMed2004

Affinity depletion of albumin from human cerebrospinal fluid using Cibacron-blue-3G-A-derivatized photopatterned copolymer in a microfluidic device.

In the context of proteomic research, affinity separations for the prefractionation of complex mixtures, such as cell lysates or human tissues, have become increasingly important. Microfluidic devices have shown significant potential to achieve fast analysis and low sample consumption. Here, we demonstrate the use of a microfluidic device to achieve affinity capture of albumin from human cerebrospinal fluid. Traditional photolithography and wet etching techniques were used to fabricate devices from borosilicate glass wafers. Monolithic porous polymer was prepared in a microfluidic channel by photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate. After derivatization with Cibacron-blue-3G-A, the modified polymer was used to achieve affinity capture of lysozyme and human albumin. Both fluorescence detection and matrix-assisted laser desorption ionization time of flight mass spectrometry were used to validate the results.

Albumins↗