1 - Introduction To Omics

Using ‘Omic Technologies
to Investigate Gene
Function
Isoelectric Focusing
• Electrophoresis across a pH gradient
• Proteins migrate to their isoelectric pH
Berg, Tymoczko & Stryer, “Biochemistry”,

6th edn, 2006, p. 73
Two-dimensional Gel
Electrophoresis
• Protein sample
initially fractionated
in one dimension by
isoelectric focusing
• SDS-PAGE
performed
perpendicular to
original direction
• Separates proteins
according to pI and
mass Berg, Tymoczko & Stryer, “Biochemistry”,
6th edn, 2006, p. 74
Two-dimensional Gel
Electrophoresis
• Proteins from E.
coli separated by
2D-electrophoresis
• >1,000 proteins
can be resolved

6th edn, 2006, p. 74
Mass Spectrometry
• MALDI-TOF mass spectrometry
• Protein sample is ionized and exposed to electrical field
• Ions travel according to size

6th edn, 2006, p. 94
MALDI-TOF Mass Spectrum
• MALDI-TOF gives good estimates of molecular weights
• Can be used to identify a few proteins within a mixture

6th edn, 2006, p. 94
Proteomic Analysis by Mass
Spectrometry
• Proteins separated by 2D
electrophoresis
• Single proteins eluted
• Digestion with trypsin
will give fragments with
unique set of sizes
• Sizes identified by mass
spectrometry and
matched to database
• Allows identification of
unknown proteins Berg, Tymoczko & Stryer, “Biochemistry”,
6th edn, 2006, p. 95
Transcriptomics v Proteomics
• Transcriptomics and proteomics are both very
powerful
• Differences in their practical application:
Transcriptomics is robust, relatively cost-
effective and user-friendly
Proteomics still relatively limited – problems can
remain with purification and stability of proteins
• Increasing potential to combine and compare data
sets - for discussion see Hegde et al., Curr. Opin.
Biotech., 14, 647-651 (2003)
Bioinformatics:
Mining the Data
Bioinformatics & Databases
 Latest biological data is gathered, organised and
disseminated through large databases
 Databases include:
- EBI, NCBI, Pfam, SMART, SWISS-PROT, TAIR
 Information in bioinformatic databases:
- sequences, structures, homology searches
 Fast search engines allow searches by all with internet access
– databases are as useful as the results they help generate!
 Improved tools for analysis of sequences
Databases – Some URLs
Resource URL
European Bioinformatics www.ebi.ac.uk/

Institute
GenBank www.ncbi.nlm.nih.gov/Genbank/
NCBI www.ncbi.nlm.nih.gov/
Protein DataBank http://www.rcsb.org/pdb/home/home.do
Sanger Centre www.sanger.ac.uk/
SMART smart.embl-heidelberg.de
The Arabidopsis www.arabidopsis.org/
Information Resource
(TAIR)
NCBI: Complete Genomes
NCBI: Eukaryotic Genomes
NCBI: Eukaryotic Genomes
NCBI: Microbial Genomes
NCBI: Microbial Genomes
Databases - The Caveats
 Databases contain mistakes (low as a proportion of

total data) -
primary data errors -
data analysis errors -
annotation errors
 Errors are difficult to correct
 Make the interpretation of data your own
responsibility!!
NCBI – Useful Links
Links to brief description of all

resources at NCBI
NCBI – Useful Links
Taxonomy: general
information about
All taxonomy
resources:
PubMed: retrieval system
provides integrated BLAST: BLAST® (Basic Local
Domains & Structure: containing citations,
access to nucleotide Alignment Search Tool) is a
Genetics & Medicine: abstracts, and indexing terms
NCBIand
Structure
proteinGroup, set of similarity search
continuously updated for journal articles in the
including
sequence tools to
data search
from programs
catalogues of human biomedical sciences
and display
>100,000 structures
organisms
genes and genetic
disorders
Databases Summary
• Many databases are available
- some have lot of general information (NCBI, EBI)
- some have specific data (Pfam, SWISS-PROT)
- some relate to specific research interests (TAIR)
• Become well acquainted with specific databases
• Wide range of databases, web sites and other
resources are available for in silico analysis of biological
data
• Great advantages, but beware caveats and potential
pitfalls – understand capabilities and limitations!
• Use information intelligently:
- always ask if the conclusions make biological sense
- may require further analyses or experimentation
“Omics” Overview
• Analyses of various biological molecules have
developed into their own scientific disciplines; e.g.
Metabolomics = “analysis of metabolites”, etc.
• Transcriptome: set of all mRNAs ("transcripts”)
produced from a genome
• Proteome: set of all proteins produced under a given
set of conditions
• Both can vary because they reflect genes that are
actively expressed at any given time
• Transcriptomics and proteomics are both powerful,
but are used differently: transcriptomics is cheaper
and more user friendly than proteomics

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Using ‘Omic Technologies

Berg, Tymoczko & Stryer, “Biochemistry”,

Berg, Tymoczko & Stryer, “Biochemistry”,

Berg, Tymoczko & Stryer, “Biochemistry”,

Berg, Tymoczko & Stryer, “Biochemistry”,

European Bioinformatics www.ebi.ac.uk/

 Databases contain mistakes (low as a proportion of

Links to brief description of all

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