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Jinzhi Chen

Publications and source records attributed to Jinzhi Chen.

8 recordsLinked to original sources
PubMed2006

A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution LC-MS.

MOTIVATION: Comparing two or more complex protein mixtures using liquid chromatography mass spectrometry (LC-MS) requires multiple analysis steps to locate and quantitate natural peptides within a single experiment and to align and normalize findings across multiple experiments. RESULTS: We describe msInspect, an open-source application comprising algorithms and visualization tools for the analysis of multiple LC-MS experimental measurements. The platform integrates novel algorithms for detecting signatures of natural peptides within a single LC-MS measurement and combines multiple experimental measurements into a peptide array, which may then be mined using analysis tools traditionally applied to genomic array analysis. The platform supports quantitation by both label-free and isotopic labeling approaches. The software implementation has been designed so that many key components may be easily replaced, making it useful as a workbench for integrating other novel algorithms developed by a growing research community. AVAILABILITY: The msInspect software is distributed freely under an Apache 2.0 license. The software as well as a Zip file with all peptide feature files and scripts needed to generate the tables and figures in this article are available at http://proteomics.fhcrc.org/.

Algorithms↗
PubMed2006

Mining the acute respiratory distress syndrome proteome: identification of the insulin-like growth factor (IGF)/IGF-binding protein-3 pathway in acute lung injury.

To obtain a more complete protein profile of the airspace milieu in acute respiratory distress syndrome (ARDS) and to identify new mediators, we analyzed bronchoalveolar lavage fluid (BALF) by shotgun proteomics. Using BALF from three patients, we identified a total of 870 different proteins, a nearly 10-fold increase from previous reports. Among the proteins identified were known markers of lung injury, such as surfactant, proteases, and serum proteins. We also identified several biologically interesting proteins not previously identified in patients with ARDS, including insulin-like growth factor-binding protein-3 (IGFBP-3). Because of the known role of IGFBP-3 in regulating cell survival, we measured IGFBP-3 levels by enzyme-linked immunosorbent assay in ARDS BALF. Normal controls had low levels of IGFBP-3, whereas patients with early ARDS had a significant increase in IGFBP-3. The IGF pathway, acting through the type 1 IGF-receptor, repressed apoptosis of lung fibroblasts but not lung epithelial cells. Furthermore, depletion of IGF in ARDS BALF led to enhanced fibroblast apoptosis. Our data suggest that the IGFBP-3/IGF pathway is involved in the pathogenesis of lung injury, illustrating the power of shotgun proteomics to catalog proteins present in complex biological fluids, such as BALF, from which hypotheses can be developed and tested.

Adult↗
PubMed2003

Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis.

An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of approximately 240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 microg. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 microm to 100 microm, the required protein loading is further decreased from 9.6 microg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.

Chromatography, Liquid↗
PubMed2003

Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry.

A miniaturized trypsin membrane reactor housed inside a commonly used capillary fitting is developed and demonstrated for enabling rapid and sensitive protein identification by on-line proteolytic digestion and analysis of protein digests using nano-ESI-MS and MALDI-MS. The design and assembly of the capillary fitting-based trypsin membrane reactor are straightforward and highly robust, without the need for expensive fabrication technology and procedures. The resultant protein digests can also be further concentrated and resolved using capillary reversed-phase liquid chromatography or transient capillary isotachophoresis/zone electrophoresis prior to the mass spectrometric analysis in an integrated platform. By comparing these results with the results obtained from our previous studies using plastic microfluidics (Gao et al., Anal. Chem. 2001, 73, 2648-2655), significant reduction in dead volume and sample consumption can be achieved using this newly developed tryptic digestion station. This nanoscale reaction system enables rapid proteolytic digestion in seconds instead of hours for a protein concentration of less than 10(-8) M, consumes very little sample (< or = 5 fmol), and offers capillary interfaces with various separation and mass spectrometry techniques. The ultrafast enzymatic turnover for attaining complete peptide coverage in protein identification is contributed by the highly porous structure of the membrane media, providing excessive trypsin loading while eliminating the constraints of diffusion-limited reaction kinetics.

Indicators and Reagents↗
PubMed2002

On-line post-capillary affinity detection of immunoglobulin G for capillary zone electrophoresis.

To address the quality issues of antibody manufacturing, post-capillary affinity detection of immunoglobulin G (IgG) is developed for capillary zone electrophoresis. In analogy to a two-dimensional separation system, capillary zone electrophoresis (CZE), as the first dimension, resolves IgG variants based on their differences in molecular structure. IgG variants separated by CZE are discriminated against other serum and cellular proteins by affinity complex formation with protein A binding fragment in a post-capillary reactor. The analytical power of post-capillary affinity detection is demonstrated for rapid and selective heterogeneity analysis of human IgG subclasses and monoclonal antibodies in complex sample matrices. By comparing with pre-capillary formation of affinity complexes between IgG and protein A, post-capillary affinity detection clearly exhibit greater resolving power for examining IgG microheterogeneity. Affinity complex formation prior to CZE analysis, however, has the advantage of lower detection limits. Detection limits suffer with post-capillary affinity detection because of the high fluorescence background contributed by the fluorescently labeled protein A in the post-capillary reactor, and the need to determine a small change in the background level upon complex formation.

Electrophoresis, Capillary↗
PubMed2002

Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation.

On-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed using a microinjector as the interface for performing two-dimensional (2-D) protein/peptide separations of complex protein mixtures. The focusing effect of CIEF not only contributes to a high-resolution protein/peptide separation, but also may permit the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. The preparative capabilities of CIEF are much larger than most of capillary-based electrokinetic separation techniques since the entire capillary is initially filled with a solution containing proteins/peptides and carrier ampholytes for the creation of a pH gradient inside the capillary. The focused peptides which have a similar pI are coinjected into the second separation dimension and further resolved by their differences in hydrophobicity. The resolving power of combined CIEF-CRPLC system is demonstrated using the soluble fraction of Drosophila salivary glands taken from a period beginning before steroid-triggered programmed cell death and extending to its completion. The separation mechanisms of CIEF and CRPLC are completely orthogonal and the overall peak capacity is estimated to be around approximately 1800 over a run time of less than 8 h. Significant enhancement in the separation peak capacity can be realized by further increasing the number of CIEF fractions and/or slowing the solvent gradient in CRPLC, however, at the expense of overall analysis time. The results of our preliminary studies display significant differences in the separation profiles of peptide samples obtained from salivary glands of animals staged at the 6 and 12 h following puparium formation.

Animals↗
PubMedDate not supplied

Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies.

Capillary isoelectric focusing (CIEF) involves the use of the entire capillary filled with a mixture containing protein/peptide analytes and carrier ampholytes. Thus, the preparative capabilities of CIEF are inherently greater than most capillary-based electrokinetic separation techniques. To further increase sample loading and, therefore, the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from a solution reservoir, is demonstrated using a low p/ protein calibration kit and tryptic peptides from Saccharomyces cerevisiae. The proteins/peptides continuously migrate into the capillary and encounter a pH gradient established by carrier ampholytes originally present in the capillary for focusing and separation. Dynamic introduction and focusing in CIEF can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Depending on the mobilities of yeast peptides, the loading capacity of each peptide is measured to be around 8 to 45-fold of that obtained in conventional CIEF. By comparing with the concentrations of dilute yeast peptides originally present in the reservoir, an overall concentration factor of 1400-7700 together with excellent separation resolution is achieved using dynamic introduction and focusing. This concentration effect is further illustrated by detecting 10 pg/microL of bradykinin peptide spiked in yeast protein digest using only ultraviolet absorption.

Chemistry Techniques, Analytical↗