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Casting Proteins on Water: 2-D Fluid Proteomics

Explore the limitations of 2-D gel electrophoresis in protein analysis and discover the benefits of a 2-D LC-MS approach for protein identification, allowing for the determination of post-translational modifications and errors in database ORF predictions.

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Casting Proteins on Water: 2-D Fluid Proteomics

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  1. #33. Cast Thy Proteins Upon the Water: Fluid Proteomics in a 2-D World Barry Moore, Chad Nelson, Mike Giddings, Mark Holmes, Melissa Kimball, Norma Wills, John Atkins, Ray Gesteland University of Utah

  2. Overview • "Standard" proteomics technique: • 2-D gel electrophoresis • Followed by MALDI-MS • But 2-D gel can't tell you full-length mass • 2-D LC-MS approach to protein identification • Simultaneously map fragments and full-length mass

  3. Uses • Determine information on: • Post-translational mods. • Acetylation • Signal peptide cleavage sites • Errors in database ORF predictions • Etc.

  4. Novel Mapping Algorithm - "MASCOT" • Does not rely on searching predefined ORFs: • In silico tryptic digest of genomic DNA • Identify regions of clustering • Thus allows identification of: • Programmed frameshifting events • Short or ambiguous ORFs missed by predictors • Applied to yeast mitochondria

  5. #306 Analyzing the Metagenome:Accessing the Uncultured Microbial World Jo Handelsman University of Wisconsin-Madison

  6. Sequencing Soil • Most studied microorganisms are cultured (!) • But most microorganisms in the environment haven't been studied for this reason • So attempt to study the "metagenome" of the soil by sequencing DNA derived directly from...a bucket of soil • Attempt to make large insert-size BAC library in hopes of identifying distinct organisms

  7. What's in there? • Discoveries made: • Insights into diversity and organization of various microorganisms • "structurally new antibiotic with broad-spectrum activity"

  8. Genome reconstruction • Thousands of species of microorganisms • Genome reassembly is quite a challenge • Starting with two model systems: • Midgut of gypsy moth larvae pH=12 (bacteria of 10 phylotypes, 6 of which are culturable) • Acid mine drainage site pH=0-1 (5 phylotypes, including Ferroplasma acidarmanus)

  9. #59 A New Apolipoprotein Influencing Plasma Triglyceride Levels in Humans and Mice Revealed by Comparative Sequence Analysis Len A. Pennacchio1, Michael Olivier3, Jaroslav A. Hubacek2, Jonathan C. Cohen2, Ronald M. Krauss1, and Edward M. Rubin1 1. Genome Sciences Department, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. 2. Center for Human Nutrition and McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas TX, 75390-9052, USA. 3. Human and Molecular Genetics Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA.

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