Yueju Wang

University of Maryland, College Park, CGS, Maryland, United States

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Publications (6)24.22 Total impact

  • Yueju Wang · Brian M Balgley · Cheng S Lee
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    ABSTRACT: The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.
    No preview · Article · Nov 2005 · Expert Review of Proteomics
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    ABSTRACT: This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high-confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 microg of total protein, an amount corresponding to 20,000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of CIEF fractions without an accompanying increase in sample consumption.
    No preview · Article · Nov 2005 · Analytical Chemistry
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    ABSTRACT: In this report, we take a heuristic approach to studying the effects of mass tolerance settings and database size on the sensitivity and specificity of MASCOT. We also examine the efficacy of the MASCOT Identity Threshold as a discriminator when applied to QqTOF data with an average mass accuracy of 10 ppm or better. As predicted, arbitrarily large mass tolerance settings negatively affect MASCOT's specificity, and to a lesser degree, sensitivity. Increased mass tolerances also render the generation of a significance threshold less effective. To study these effects, we used Bayes' Law to calculate MASCOT's predictive values. With a relatively small search database (Human IPI), MASCOT had a mean positive predictive value of 0.993 when combined with MASCOT's Identity Threshold. However, the corresponding average negative predictive value, or the probability that an ion was not present given no score or a score below threshold, was reduced as mass tolerances were tightened, and had an average value of 0.717. This value was improved upon by extrapolating an empirical threshold using a reversed database search and a new algorithm to rapidly identify false positive identifications. Using the empirical threshold reduced false negative identifications on the average 17% while limiting the false positive rate to below 5%; even larger reductions were obtained using mass tolerances approaching two times the actual error of the experimental data. A simple application of this strategy to the analysis of a microdissected glioblastoma multiforme sample analyzed by IEF/LC-MS/MS is reported, as is a description of the tools required to implement a large scale analysis using this alternative approach.
    No preview · Article · Aug 2005 · Journal of Proteome Research
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    ABSTRACT: For top-down proteomics, nano-reversed phase liquid chromatography (RPLC) plays a major role in both single and multidimensional protein separations in an effort to increase the overall peak capacity for the resolution of complex protein mixtures prior to mass spectrometry analysis. Effects of various chromatography conditions, including alkyl chain length in the stationary phase, capillary column temperature, and ion-pairing agent, on the resolution of intact proteins are studied using nano-RPLC-electrospray ionization-mass spectrometry. Optimal chromatography conditions include the use of C18 column heated at 60 degrees C and the addition of trifluoroacetic acid instead of heptafluorobutyric acid as the ion-paring agent in the mobile phase. Under optimized chromatography conditions, there are no significant differences in the separation performance of yeast cell lysates present in the native versus denatured states. Denatured yeast proteins resolved and eluted from nano-RPLC can be subjected to proteolytic digestion in an on- or off-line approach to provide improved protein sequence coverage toward protein identification in a combined top-down/bottom-up proteome platform.
    No preview · Article · Jun 2005 · Journal of Chromatography A
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    ABSTRACT: An integrated protein concentration/separation platform, combining capillary isoelectric focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction "parked" in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry.
    No preview · Article · Feb 2005 · Journal of Proteome Research
  • Jonathan W Cooper · Yueju Wang · Cheng S Lee
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    ABSTRACT: The sequencing of several organisms' genomes, including the human's one, has opened the way for the so-called postgenomic era, which is now routinely coined as "proteomics". The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists of protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Still, the 2-D PAGE-mass spectrometry (MS) approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses as well as for membrane proteins), dynamic range, sensitivity, and throughput. Consequently, considerable efforts have been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings in 2-D PAGE while reserving the ability to resolve complex protein and peptide mixtures prior to MS analysis. This review focuses on the most recent advances in capillary-based separation techniques, including capillary liquid chromatography, capillary electrophoresis, and capillary electrokinetic chromatography, and combinations of multiples of these mechanisms, along with the coupling of these techniques to MS. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated proteome effort. Miniaturized and integrated multidimensional peptide/protein separations using microfluidics are further summarized for their potential applications in high-throughput protein profiling toward biomarker discovery and clinical diagnosis.
    No preview · Article · Dec 2004 · Electrophoresis