Showing papers by "Jimmy K. Eng published in 2000"

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry.

Abstract: Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (μLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyamidomethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interf...

Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation.

Abstract: The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.

Mass spectral investigations on microorganisms

Abstract: Bacterial cells undergo lysis readily, when suspended in mild aqueous acids, and release the cellular proteins along with other biomolecules. Molecular masses of the protein biomarkers released in-situ from individual intact bacterial cells could be directly measured by mass spectrometry. Limited sample clean up may be required at times, prior to mass spectral analysis, to remove any ionizable impurities such as salts, buffers and deergents. The marker proteins specific for individual genus, species and strains were determined by the comparison of the biomarkers measured for several closely related organisms. Even though there is a probability of over 4000 cellular proteins expressed in any single bacterial cell, only a small fraction of the projected marker proteins are identified consistently during the process. This could be due to the variation in the ionization properties of the proteins and the limited energy available to prompt their ionization. Variation in the sample processing and culture condit...

Direct Analysis of Protein Complexes

Abstract: Deciphering the functions of genes discovered by genome sequencing will be a major challenge of the post-genome era. Physiological processes are performed primarily by proteins and these functions are often accomplished with other proteins as components of multi-protein complexes, as part of signal transduction pathways or as ligands for receptors. Identifying the sets of proteins involved in a process will be key to understanding the individual roles of proteins. Approaches to accomplish this goal will encompass at least two types of measurements. First, by measuring expression levels of proteins under different cellular conditions and states, co-regulation of protein expression can be observed. Co-regulation will not necessarily indicate proteins are part of a complex or pathway, but that their functions are regulated as part of the process. A second measurement will encompass dissection of the components of protein complexes. Proteins perform many of their functions in concert with other proteins, by forming stable complexes or through more subtle interactions. A protein’s presence in a complex is more reflective of direct involvement in a process then association by co-regulation. Some proteins in complexes may be co-regulated and others may not be. By identifying the interacting proteins as a function of cellular state, insight into the networks of proteins involved in those processes will be obtained.