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Proteomics
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Lecture notes on Proteomics
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What is the human genome and proteome?The exact number of human genes and proteins is still being
debated
1. The GENCODE gene set (EBI):
a. 19,901 protein-coding genes
b. 15,779 non-coding genes.
2. RefSeq, (NCBI):
a. 20,203 protein coding genes
b. 17,871 non-coding genes
Variation in protein coding gene number
- Inconsistency in data bases
- You only get what you search for
- Manual curation v. computer
Proteome complexity
Proteomics is the large-scale study of proteins. Identification
and analysis of the protein content in a cell, tissue, organisms
- Functional proteomics v. entire proteome
- Requires and enhances genome information
- "Snapshot" of the protein environment – DISEASE v.
HEALTHY
- Major advancement: mass spectrometry
Proteomics provides information on:
- Protein isoforms
- Differential gene expression
- Spatiotemporal dynamics
- Genomic variation
- Post-translational modifications
- Protein interaction with other proteins, nucleic acid
and/or metabolites
- Cellular localization of proteins
, - Protein stability
- translational and post-translational levels of gene-expression regulation
Proteomics techniques - 2-dimensional gel electrophoresis (2D-GE)
1st Dimension: Isoelectric focusing tube gel. Separates proteins according to
their isoelectric point, where their net charge is zero compared to the pH of the
gel.
2nd Dimension: Rotate tube gel 90° and place onto SDS-polyacrylamide gel
(SDS-PAGE). Separates proteins according to molecular weight
Advantages of 2D-GE:
- Resolves hundreds of proteins
- Identifying new proteins
- Measure their relative abundance
Disadvantages of 2D-GE:
- Challenging spot-to-spot comparison/matching
- Proteins do not migrate to the same point on each gel (IEF or SDS-PAGE)
- Need for computer-based spot matching
- Small errors could prevent meaningful quantitation
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