Transcript Bioinformatics and Functional Genomics, Chapter 8, Part 1

Protein Structure &
Analysis- part 2
Biology 224
Dr. Tom Peavy
Sept 27 & 29
<Images from Bioinformatics and Functional
Genomics by Jonathan Pevsner>
Gene Ontology (GO) Consortium
The Gene Ontology Consortium
An ontology is a description of concepts. The GO
Consortium compiles a dynamic, controlled vocabulary
of terms related to gene products.
There are three organizing principles:
Molecular function
Biological process
Cellular component
GO terms are assigned to Entrez Gene entries
Page 241
Example
Gene product cytochrome c GO entry terms:
molecular function = electron transporter activity,
the biological process = oxidative phosphorylation and
induction of cell death
the cellular component = mitochondrial matrix and
mitochondrial inner membrane.
GO consortium (http://www.geneontology.org)
No centralized GO database. Instead, curators
of organism-specific databases assign GO terms
to gene products for each organism.
AmiGO is the searchable portion of the GO
--Gene Symbol, name, UniProt access numbers, and
Text searches can be used to find GO entries
The Gene Ontology Consortium: Evidence Codes
IC
IDA
IEA
IEP
IGI
IMP
IPI
ISS
NAS
ND
TAS
Inferred by curator
Inferred from direct assay
Inferred from electronic annotation
Inferred from expression pattern
Inferred from genetic interaction
Inferred from mutant phenotype
Inferred from physical interaction
Inferred from sequence or structural similarity
Non-traceable author statement
No biological data
Traceable author statement
ProDom entry for HIV-1 pol shows many related proteins
Page 231
Physical properties of proteins
Many websites are available for the analysis of
individual proteins. ExPASy and ISREC are two
excellent resources.
The accuracy of these programs is variable.
Predictions based on primary amino acid sequence
(such as molecular weight prediction) are likely to be
more trustworthy. For many other properties (such as
posttranslational modification of proteins by
specific sugars), experimental evidence may be
required rather than prediction algorithms.
Page 236
Access a variety of protein analysis programs
from the top right of the ExPASy home page
Page 235
Page 244
Page 244
Proteomics: High throughput protein analysis
Proteomics is the study of the entire collection
of proteins encoded by a genome
“Proteomics” refers to all the proteins in a cell
and/or all the proteins in an organism
Large-scale protein analysis
2D protein gels
Yeast two-hybrid
Rosetta Stone approach
Pathways
Page 247
Two-dimensional protein gels
First dimension: isoelectric focusing
Second dimension: SDS-PAGE
Page 248
Two-dimensional protein gels
First dimension: isoelectric focusing
Electrophorese ampholytes to establish
a pH gradient
Can use a pre-made strip
Proteins migrate to their isoelectric point
(pI) then stop (net charge is zero)
Range of pI typically 4-9 (5-8 most common)
Page 248
Two-dimensional protein gels
Second dimension: SDS-PAGE
Electrophorese proteins through an acrylamide
matrix
Proteins are charged and migrate through an
electric field
Conditions are denaturing (SDS)
and reducing (2-mercaptoethanol)
Can resolve hundreds to thousands of proteins
Page 248
Proteins identified on 2D gels (IEF/SDS-PAGE)
Direct protein microsequencing by
Edman degradations
-- done at many core facilities (e.g. UC Davis)
-- typically need 5 picomoles
-- often get 10 to 20 amino acids sequenced
Protein mass analysis by MALDI-TOF
-- done at core facilities
-- often detect posttranslational
modifications
-- matrix assisted laser desorption/ionization
time-of-flight spectroscopy
Page 250-1
Page 252
Evaluation of 2D gels (IEF/SDS-PAGE)
Advantages:
Visualize hundreds to thousands of proteins
Improved identification of protein spots
Disadvantages:
Limited number of samples can be processed
Mostly abundant proteins visualized
Technically difficult
Page 251