Search PubMed⌕ Search

Biomedical subjects

Yu-Chie Chen

Publications and source records attributed to Yu-Chie Chen.

At least 19 recordsLinked to original sources
PubMed2007

Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis.

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach.

Aluminum Oxide↗
PubMed2006

Detection of phosphopeptides by localized surface plasma resonance of titania-coated gold nanoparticles immobilized on glass substrates.

We herein demonstrate a new sensing method for phosphopeptides by localized surface plasmon resonance (LSPR) using titania-coated gold nanoparticles immobilized on the surface of a glass slide as the sensing substrate and using UV-visible spectrophotometry as the detection tool. Titania has been known to be an effective substrate for binding with phosphorylated species. The detection principle is the shift of wavelength of optical absorption due to SPR of the gold nanoparticles induced by binding of phosphorylated species with titania on the surface of the gold nanoparticles. The feasibility of the approach is demonstrated by detection of tryptic digest products of beta-casein and milk. Gold nanoparticles coated with thin films of titania, immobilized on a glass slide, can selectively bind traces of phosphopeptides from complex samples, resulting in a wavelength shift of the absorption band in the SPR spectrum with good reproducibility. The LSPR results are confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The detection limit for the tryptic digest product of beta-casein is 50 nM.

Glass↗
PubMed2006

Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fraction.

A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for approximately 3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal.

Animals↗
PubMed2006

Affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for SALDI MS analysis of peptides and proteins.

Silane-immobilized magnetic iron oxide particles were used as the assisting material in surface-assisted laser desorption/ionization (SALDI) mass spectrometric analysis. This approach can be used to analyze small proteins and peptides. The upper detectable mass range is approximately 16 kDa. The detection limit for peptides is about 20 fmol. Silanized iron oxide particles with negatively charged functionalities can also be used as the affinity probes to selectively trap oppositely charged species from sample solutions by adjusting the pH of the solution. A tryptic digest product of cytochrome C at a concentration as low as 10 nM can be enriched by the particles and directly analyzed by iron oxide SALDI MS without the need for elution steps. Affinity-based mass spectrometry using the bifunctional silanized magnetic iron oxide particles as the SALDI matrix and concentrating probe is demonstrated in this study.

Ferric Compounds↗
PubMed2006

Determination of calcium in complex samples using functional magnetic beads combined with electrodeless/sheathless electrospray ionization mass spectrometry.

Conventionally, electrospray ionization ion trap mass spectrometry (ESI-ITMS) is not used for the analysis of metal ions because metal ions may be of too low mass to be trapped by the mass analyzer. Furthermore, metal ions can easily precipitate during ESI processes because of their poor volatilities. We present an approach for solving these two problems using milk as the sample to demonstrate the feasibility of using ESI-ITMS as the detection method for metal ions. Iron oxide magnetic beads with nitrilotriacetic acid (NTA) immobilized on their surface were used as affinity probes for calcium ions from dairy drinks. Ethylenediaminetetraacetic acid (EDTA) was used for the elution of the metal ions chelated by NTA on the beads. The EDTA eluent was analyzed by electrodeless/sheathless ESI-ITMS. The use of functional magnetic beads to trap metal ions from complex samples can effectively reduce the interference from sample matrix. Using the EDTA eluent as the sample avoids the problem of the low volatility of metal ions during ESI-ITMS analysis. This method gives measurements of calcium ions in dairy drinks quantitatively very close to the true values (<4% error).

Animals↗
PubMed2005

Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry.

Columns packed with microsized titanium dioxide particles have been used effectively as precolumns for enriching phosphopeptides from complex mixtures. Nanosized titanium dioxide particles have a higher specific surface area and, hence, potentially higher trapping capacities toward phosphopeptides than do microsized particles. Thus, in this study, we employed TiO2-coated magnetic (Fe3O4/TiO2 core/shell) nanoparticles to selectively concentrate phosphopeptides from protein digest products. Because of their magnetic properties, the Fe3O4/TiO2 core/shell nanoparticles that are conjugated to the target peptides can be isolated readily from the sample solutions by employing a magnetic field. In this paper, we also demonstrate that the Fe3O4/TiO2 core/shell nanoparticles behave as an effective SALDI matrix: our upper detectable mass limit was approximately 24 000 Da, whereas the detection limit for peptides was in the low-femtomole range. That is to say, the target analytes trapped by the Fe3O4/TiO2 nanoparticles can be identified by introducing the particles directly into the mass spectrometer for TiO2-SALDI-MS analysis without the need for any further treatment. For example, elution steps are not necessary when using this approach. In addition, the trapping selectivity of these Fe3O4/TiO2 nanoparticles toward phosphopeptides was quite good. These properties combine to result in the low detection limits. The lowest detectable concentration of phosphopeptides that we analyzed using this approach was 500 pM for a 100-microL tryptic digest solution of beta-casein; this level is much lower than that which can be obtained using any other currently available method.

Amino Acid Sequence↗
PubMed2005

Sheathless capillary electrophoresis/electrospray ionization mass spectrometry using a pulled bare fused-silica capillary as the electrospray emitter.

It has always been assumed that electrical contact at the capillary outlet is a necessary requirement when coupling capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). In this study, we used a pulled bare-capillary tip as the ESI emitter, but neither was it coated with any electrically conductive materials nor was a high external voltage applied on its outlet. In this paper, we demonstrate that this straightforward approach may be used to generate multiply charged ions of proteins and peptides through electrospray ionization. Our results indicate that peptides and proteins, including bradykinin, cytochrome c, myoglobin, and tryptic digest products that elute from a pulled bare-capillary tip can be detected directly by ESI-MS using the tapered bare-capillary interface. Thus, we have demonstrated that CE and ESI-MS may be combined successfully without the need to modify the outlet of the capillary tip with an electrically contacting material.

Journal Article↗
PubMed2005

Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides a straightforward means to differentiate microorganism species based on mass spectral fingerprinting. The pathogen cell concentration in an infected sample, however, is generally lower than that capable of being detected directly by MALDI-MS. Furthermore, the presence of proteins or metabolites in biological fluids always causes unavoidable interference for the identification of microorganism species. Vancomycin, which binds to D-Ala-D-Ala moieties on the cell walls of Gram-positive bacteria and, therefore, inhibits peptidoglycan synthesis, is one of the most potent antibiotics. Thus, we have employed vancomycin-modified magnetic nanoparticles as affinity probes to selectively trap Gram-positive pathogens from sample solutions; i.e., these bacteria can be isolated from sample solutions by applying a magnetic field. The isolated cells could then be characterized by MALDI-MS. This approach effectively reduces the interference of protein and metabolite signals in the mass spectra of Gram-positive bacteria because vancomycin has such high specificity for the D-Ala-D-Ala units of the cell walls. The lowest cell concentration we detected for both Staphylococcus saprophyticus and Staphylococcus aureus in a urine sample (3 mL) was approximately 7 x 10(4) cfu/mL.

Cell Wall↗
PubMed2005

Characterization of intact Penicillium spores by matrix-assisted laser desorption/ionization mass spectrometry.

The fungal spores of Penicillium expansum, P. chrysogenum, P. citrinum, P. digitatum, P. italicum, and P. pinophilum were characterized by using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOFMS). These fungal spores are frequently found in grain and fruit. The mass spectra of these six species were directly obtained from the intact spores without any pretreatment. The results obtained indicate that 2,5-dihydroxybenzoic acid and sinapinic acid are suitable matrices for the analysis of Penicillium spores. Characteristic ions representing the different species were obtained with sufficiently high reproducibility that these ions can be employed to identify the different fungal species. On the basis of these characteristic ions obtained from these authentic Penicillium spores, the approach was applied to characterize the fungal species contaminating the surfaces of fruit. It was demonstrated that the fungal spores directly scratched from the surfaces of fruit contaminated by unknown fungi can be rapidly identified using MALDI-TOFMS analysis without any tedious pretreatment.

Food Contamination↗
PubMed2004

Using biofunctionalized nanoparticles to probe pathogenic bacteria.

In this paper, we report a method for fabricating biofunctionalized nanoparticles by attaching human immunoglobulin (IgG) onto their surfaces through either electrostatic interactions or covalent binding. We found that these IgG-presenting nanoparticles can bind selectively to the cell walls of pathogens that contain IgG-binding sites based on the investigation of transmission electron microscopy images. Our results demonstrate that such Au-IgG nanoparticles may serve as useful nanoscale probes for exploring the interactions between IgG and pathogens. Furthermore, the IgG-presenting magnetic nanoparticles have been employed as effective affinity probes for selectively concentrating traces of target bacteria from sample solutions. The trapped bacteria were then characterized by using matrix-assisted laser desorption/ionization mass spectrometry. The lowest cell concentration we detected for both Staphylococcus saprophyticus and Staphylococcus aureus in aqueous sample solutions (0.5 mL) was approximately 3 x 10(5) cfu/mL, while the detectable cell concentration for S. saprophyticus in a urine sample was approximately 3 x 10(7) cfu/mL.

Bacterial Proteins↗
PubMed2004

Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis.

MALDI mass spectrometry is used widely in various fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range, but suppression effects between analyte molecules and interference from the sample matrix frequently arise during MALDI analysis. The suppression effects can be avoided if target species are isolated from complicated matrix solutions in advance. Herein, we proposed a novel method for achieving such a goal. We describe a strategy that uses gold nanoparticles to capture charged species from a sample solution. Generally, ionic agents, such as anionic or cationic stabilizers, encapsulate gold nanoparticles to prevent their aggregation in solution. These charged stabilizers at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. We have employed this concept to develop nanoparticle-based probes that selectively trap and concentrate target species in sample solutions. Additionally, to readily isolate them from solution after attracting their target species, we used gold nanoparticles that are adhered to the surface of magnetic particles through S-Au bonding. A magnet can then be employed to isolate the Au@magnetic particles from the solution. The species trapped by the isolated particles were then characterized by MALDI MS after a simple washing. We demonstrate that Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins from aqueous solutions. In addition, we have employed Au@magnetic particle-based probes successfully to concentrate low amounts of peptide residues from the tryptic digest products of cytochrome c (10(-7) M).

Amino Acid Sequence↗
PubMed2004

Molecularly imprinted TiO2-matrix-assisted laser desorption/ionization mass spectrometry for selectively detecting alpha-cyclodextrin.

This study describes a new means to conduct molecular recognition-based analysis using mass spectrometry. Taking advantage of the unique characteristic of the absorption capacity of the TiO(2) sol-gel material in the UV region, a TiO(2) sol-gel-deposited thin film was employed as the sample substrate to assist in UV laser desorption/ionization of analytes. Sol-gels are polymeric materials that are easy to prepare and modify at low temperatures. Molecularly imprinted TiO(2) sol-gels were generated for molecular recognition-based analysis. alpha-Cyclodextrin (CD) was selected as the template molecule and doped into TiO(2) in a sol-gel reaction. The molecularly imprinted TiO(2) sol was spin-coated on a glass slide, and appropriate template cavities in the TiO(2) sol-gel material were formed after the template molecules were removed. We demonstrate that this modified glass slide can be used to select alpha-CD from a sample solution containing equal amounts of alpha-, beta-, and gamma-CD (50 ppb each, 18 mL); alpha-CD was directly detected from the modified glass slide by matrix-assisted laser desorption/ionization mass spectrometry without the addition of extra matrix. This approach provides a new detection method for molecular recognition-based analysis.

Bradykinin↗
PubMed2004

Glass-chip-based sample preparation and on-chip trypic digestion for matrix-assisted laser desorption/ionization mass spectrometric analysis using a sol-gel/2,5-dihydroxybenzoic acid hybrid matrix.

A glass-chip-based sample preparation method for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of tryptic digests of proteins and intact cells is described. A MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), was hybridized with sol-gels to generate a sol-gel-derived material. Taking advantage of the characteristics of sol-gels, the sol-gel-derived material readily adhered to the surface of a glass chip through covalent bonding. Only one step of sample preparation, deposition of the sample solution on the glass chip, was required before MALDI-MS analysis. Because 2,5-DHB was homogeneously dispersed on the sol-gel network structure, good spot-to-spot reproducibility was obtained in MALDI analysis using this approach and the analyte signals were uniform throughout the chip. The modified glass chips were robust and effective for at least 1 week. This glass-chip-based matrix preparation method provides a straightforward approach to developing techniques for analyzing the on-chip enzymatic digestion of proteins and intact cells of microorganisms. Cytochrome C and Escherichia coli were used as analytes to demonstrate the feasibility of this approach. The products of the on-chip enzymatic digests were identified through protein database searches.

Amino Acid Sequence↗
PubMed2004

Using sol-gel/crown ether hybrid materials as desalting substrates for matrix-assisted laser desorption/ionization analysis of oligonucleotides.

This study demonstrates the feasibility of using sol-gel/crown ether hybrid materials as sample substrates that reduce the intensity of the signals of sodium ion adducts of oligonucleotides during matrix-assisted laser desorption/ionization (MALDI) analysis. 2-Hydroxymethyl[15]crown-5 and 2-hydroxymethyl[18]crown-6 were added as dopants during the sol-gel process to generate desalting substrates for MALDI sample deposition. The results demonstrate that the sol-gel/crown ether hybrid materials effectively suppress the formation of sodiated oligonucleotides during MALDI analysis. The largest detectable molecular size for an oligonucleotide was a 100-mer, and the detection limit for an oligonucleotide 36-mer was ca. 20 fmol.

Crown Ethers↗
PubMed2004

Desorption/ionization mass spectrometry on nanocrystalline titania sol-gel-deposited films.

This paper describes a matrix-free method for performing desorption/ionization directly from mesoporous nanocrystalline titania sol-gel thin films, which have good absorption capacity in the ultraviolet (UV) range and can act as assisting materials during UV matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. A high concentration of citrate buffer was added into this system to provide the proton source and to reduce the presence of alkali cation adducts of the analytes. The analyte signals appear uniformly over the whole sample deposition area. Protonated molecules (MH(+) ions) of analytes dominate the titania MALDI mass spectra. Surfactants, peptides, tryptic digest products, and small proteins with molecular weights below ca. 24 000 Da, are observed in the titania MALDI mass spectra. Detection limits for insulin are as low as ca. 2 fmol with mass resolution of ca. 660.

Bradykinin↗
PubMed2004

Carbon nanotubes as affinity probes for peptides and proteins in MALDI MS analysis.

Recently, carbon nanotubes (CNTs) have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191). In a somewhat related study, we have employed CNTs produced by using NaH-treated anodic aluminum oxide (Na@AAO) as a reactive template as the assisting matrix for MALDI analysis upon the addition of high concentrations of citrate buffer. Our results indicate that the mass range can be extended to ca. 12,000 Da and that alkali metal adducts of analytes are effectively reduced. Furthermore, we have employed citric acid-treated CNTs as affinity probes to selectively concentrate traces of analytes from aqueous solutions. High concentrations of salts and surfactants in the sample solutions are also tolerated. This approach is very suitable for the MALDI analysis of small proteins, peptides, and protein enzymatic digest products.

Affinity Labels↗
PubMed2003

Reducing the alkali cation adductions of oligonucleotides using sol-gel-assisted laser desorption/ionization mass spectrometry.

The alkali cation adductions of oligonucleotides dramatically degrade MALDI mass spectra and even affect the detection limit. Desalting is generally involved in MALDI sample preparation. This work demonstrates the feasibility of using 3,4-diaminobenzoic acid (DABA) and 3,5-DABA as the MALDI matrix for oligonucleotide analysis. Furthermore, sodium ion adducts of oligonucleotides were simultaneously reduced in the mass spectra when DABA was used as the MALDI matrix and sol-gel material was used as the sample support. However, depositing the sample on the sample support was very difficult, and the lack of homogeneity of analytes/matrix distribution on the sample support also led the analyte signals to be revealed only in "sweet spots". Alternatively, DABA was doped into sol-gel materials to generate homogeneous DABA/sol-gel hybrid film. The DABA/sol-gel hybrid film was used as the sample substrate to assist the desorption/ ionization of analytes. The analyte signals were evenly found on the sample substrate. The sodium ion adductions of oligonucleotides were also effectively suppressed. The sample preparation used in this approach resembles that used in the authors' previous study, involving sol-gel-assisted laser desorption/ionization (SGALDI) mass spectrometry (Lin, Y.-S.; Chen, Y.-C. Anal Chem. 2002, 74, 5793-5798.) The SGALDI approach was demonstrated to be effective in assisting the desorption/ionization of peptides and small proteins. Herein, the SGALDI material, DABA/sol-gel hybrid material, was successfully applied to oligonucleotide analysis, and good-quality mass spectra were obtained without extra desalting. Additionally, the presence of 0.1% SDS in the oligonucleotide sample solution was tolerated without degrading the mass spectra. The largest detectable molecular size for oligonucleotides was 72 mer. The detection limit for 24 mer of oligonucleotide was 20 fmol.

Aminobenzoates↗