Biomedical subjects

Wei-Jun Qian

Publications and source records attributed to Wei-Jun Qian.

30 records · Page 2Linked to original sources

PubMed2004

High-throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology.

A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves (18)O labeling of tryptic peptides, high-efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of naïve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis. Advantages of this technology include the following: (1) a simple, highly efficient method for enriching cysteinyl-peptides; (2) a high-throughput strategy suitable for extensive proteome analysis; and (3) improved labeling efficiency for better quantitative measurements. This technology enhances both the functional analysis of biological systems and the detection of potential clinical biomarkers.

Cells, Cultured↗

PubMed2004

High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry.

The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.

Animals↗

PubMed2004

Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islets and islet cells by fluorescence microscopy.

A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at beta-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+ secretion coupling and any application, requiring rapid assays of secretion.

Animals↗

PubMed2003

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Amino Acid Sequence↗

PubMed2003

Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events.

Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.

Animals↗

PubMed2003

Microscale determination of purines in tissue samples by capillary liquid chromatography with electrochemical detection.

A microscale method for purines involved in intracellular signaling and energy metabolism, including ADP, ATP, cyclic-AMP, NADH and GTP, was developed. The analytes were separated on a fused-silica capillary liquid chromatography column (50 microm inner diameter by 25 cm long) packed with 7 microm reversed-phase particles and detected with a carbon fiber cylinder microelectrode at +1.50 V versus Ag/AgCl reference electrode. With an acetonitrile gradient, the separation was carried out within 15 min. With a 100 nl injection volume, the detection limits varied from 0.9 to 8 fmol depending upon the analyte. The low detection limits make the method suitable for analysis of small tissue samples. As a demonstration of the method, islets of Langerhans were analyzed for their adenosine-related messenger content.

Animals↗

PubMed2002

Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator.

A novel Zn2+-selective visible wavelength fluoroionophore (FluoZin-3, 9) was synthesized. The chelating portion of the molecule resembles known EGTA-based Ca2+-selective fluoroionophores, except that one of the N-acetic acid moieties has been deleted in 9. FluoZin-3 is virtually non-fluorescent in the absence of Zn2+, and exhibits a several hundred-fold fluorescence increase upon saturation with Zn2+( approximately 100 nM), with a Kd = 15 +/- 2 nM. A 1:1 binding stoichiometry of 9:Zn2+ was determined, and the fluorescence of the complex is pH-independent at pH > 6. FluoZin-3 was used to monitor Zn2+ that was co-secreted with insulin from pancreatic beta-cells by exocytosis following stimulation with glucose. The total Zn2+ concentration near the cells reached 600 nM, and Zn2+ was detectable at least 15 mum away from secreting cells. Heterogeneity in secretion among cells was indicated in that some cells in a cluster did not release Zn2+. Also, within secreting cells some regions of the cell membrane gave rise to secretion while others did not, suggesting active zones of secretion on the cell surface.

Cations, Divalent↗

PubMedDate not supplied

Advances in proteomics data analysis and display using an accurate mass and time tag approach.

Proteomics has recently demonstrated utility for increasing the understanding of cellular processes on the molecular level as a component of systems biology approaches and for identifying potential biomarkers of various disease states. The large amount of data generated by utilizing high efficiency (e.g., chromatographic) separations coupled with high mass accuracy mass spectrometry for high-throughput proteomics analyses presents challenges related to data processing, analysis, and display. This review focuses on recent advances in nanoLC-FTICR-MS-based proteomics approaches and the accompanying data processing tools that have been developed to display and interpret the large volumes of data being produced.

Animals↗

PubMedDate not supplied

Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome.

Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC-MS/MS analyses. The MS/MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, suggesting that the false positive rates are significantly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039-1048), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242-247). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications from mammalian proteomes without greatly decreasing the number of identifications.

Blood Proteins↗

PubMedDate not supplied

Utilizing human blood plasma for proteomic biomarker discovery.

Candidate proteomic biomarker discovery from human plasma holds both incredible clinical potential as well as significant challenges. The dynamic range of proteins within plasma is known to exceed 10(10), and many potential biomarkers are likely present at lower protein abundances. At present, proteomic based MS analyses provide a dynamic range typically not exceeding approximately 10(3) in a single spectrum, and approximately 10(4)-10(6) when combined with on-line separations (e.g., reversed-phase gradient liquid chromatography), and thus are generally insufficient for low level biomarker detection directly from human plasma. This limitation is providing an impetus for the development of experimental methodologies and strategies to increase the possible number of detections within this biofluid. Discussed is the diversity of available approaches currently used by our laboratory and others to utilize human plasma as a viable medium for biomarker discovery. Various separation, depletion, enrichment, and quantitative efforts as well as recent improvements in MS capabilities have resulted in measurable improvements in the detection and identification of lower abundance proteins (by approximately 10-10(2)). Despite these improvements, further advances are needed to provide a basis for discovery of candidate biomarkers at very low levels. Continued development of depletion and enrichment techniques, coupled with improved pre-MS separations (both at the protein and peptide level) holds promise in extending the dynamic range of proteomic analysis.

Biomarkers↗

PubMedDate not supplied

Development and evaluation of a micro- and nanoscale proteomic sample preparation method.

Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 microg protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (approximately 30 microg total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells (approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.

Animals↗

PubMedDate not supplied

Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

Amino Acid Motifs↗