Transcript Proteomics Applications - Genetics 564 redirect page

Mass spectrometry and
proteomics
Mass spectrometry and
proteomics
Eva Dimitrova and Jessica Connor
The “omics” nomenclature…
Genomics
DNA (Gene)
Transcription
Transcriptomics
RNA
Translation
Functional
Genomics
Proteomics
PROTEIN
Enzymatic
reaction
Metabolomics
METABOLITE
Proteomics definition
“Proteomics is a science that focuses
on the study of proteins : their roles,
their structures, their localization, their
interactions, and other factors.”
www.lexicon-biology.com
3 Kinds of Proteomics
• Functional Proteomics
The identification of protein functions, activities or
interactions at a global or organismwide scale
• Expressional Proteomics
The analysis of global or organismwide changes in protein
expression
• Structural Proteomics
The high throughput, or high volume expression and structure
determination of proteins by Xray, NMR or computerbased
methods
Components of
Expressional Proteomics
Protein Separation
Mass Spectroscopy
Bioinformatics
Pathway
Step 1: Sample prep
Step 2: Separation
Step 3: Mass spectrometry
Movie
General overview
Aebersold, R & Mann, M. (2003,
March). Mass spectrometry based
proteomics. Nature. 422, 198-207
Sample preparation
Sample preparation
•
•
•
•
Sample preparation involves everything that
lies between the sample and 1st dimension of
the 2D SDS gel
Cells and cell cultures – multiply
Homogenation and protein isolation
Contaminant removal/ cleanup
Fractionation
Cleanup and fractionation
• General Purpose Cleanup
• Improve Resolution
• Improve Reproducibility
• Fractionation
• Reduce Complexity
• Improve Range of Detection
• Enrich low-abundance proteins
www.expressionproteomics.com
Separation
2D-SDS PAGE gel
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
The second dimension
(separation by mass)
-pH gel strip is loaded onto a SDS gel
-SDS denatures the protein (to make
movement solely dependent on mass,
not shape) and eliminates charge.
Can Resolve: ~1500-2500 proteins
Staining Technology
• Staining
–Silver
–Coomassie blue
–Fluorescent dyes
•Sypro Ruby-$$$
–Radioisotopic labeling
Trypsin digestion
Trypsin
• Serine protease
• Claves at the carboxyl end of lysine and
arginine (except when either is followed by
proline)
Mass Spectrometry
How does a mass spectrometer work?
Create ions
Separate ions
• Ionization
method
• Mass analyzer
– MALDI
– Electrospray
(Proteins must be
charged and dry)
– MALDI-TOF
– Quadrapole
– MALDI-QqTOF
•
AA seq and MW
Detect ions
• Mass
spectrum
• Database
analysis
– QqTOF
•
AA seq and protein modif.
Definitions
ESI- Electron Spray Ionization
is a technique used in mass spectrometry to produce ions. It is
especially useful in producing ions from macromolecules
because it overcomes the propensity of these molecules to
fragment when ionized
MALDI- Matrix-assisted laser desorption/ionization
is a soft ionization technique used in mass spectrometry,
allowing the analysis of biomolecules and large organic
molecules, which tend to be fragile and fragment when ionized
by more conventional ionization methods.
Mass analyser
• TOF – time of flight
• Ion trap
• Quadropole
• Fourier transform ion cyclotron
Mass Spec Principles
Sample
+
_
Ionizer
Mass Analyzer
Detector
Typical Mass Spectrum
Relative Abundance
aspirin
m/z ratio:
Molecular weight
divided by the charge
on this protein
120 m/z-for singly charged ion this is the mass
ESI and MALDI
Aebersold, R & Mann, M. (2003, March).
Mass spectrometry based proteomics.
Nature. 422, 198-207
Peptide sample
Stable isotope protein labeling
Aebersold, R & Mann, M. (2003, March). Mass
spectrometry based proteomics. Nature. 422,
198-207
Peptide Mass Identification
Library
Spot removed
from gel
Artificial
spectra built
Fragmented
using trypsin
Spectrum of
fragments
generated
MATCH
Artificially
trypsinated
Database of
sequences
(i.e. SwissProt)
How MS
sequencing works
• Peptide mass and database
matching
• Further f ragmentation of the
peptides occur in a predictable
fashion, mainly at the peptide bonds
• The resulting daughter ions have
masses that are consistent with
KNOWN molecular weights of dipeptides, tri-peptides, tetrapeptides…
Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc…
Peptide sample
Peptide Hits
Data Analysis Limitations
-You are dependent on well annotated genome
databases
-Data is noisy. The spectra are not always
perfect. Often requires manual determination.
-Database searches only give scores. So if you
have a false positive, you will have to manually
validate them
Proteomics Applications
Why Proteomics?
• Proteins are the active biological agents in
cells
• DNA sequences don’t show how proteins
function or how biological processes occur
• Proteins undergo post transcriptional
modifications
• 3D structures affect protein function
• Alternative splicing
The Human Proteome Initiative. (2007) http://ca.expasy.org/sprot/hpi/hpi_desc.html
Retrieved March 24, 2009.
Challenges
• Analyses of complex mixtures are not
comprehensive
• Difficult to prepare a pure sample
• Protein expression is very sensitive to
environmental conditions
• Difficult to use ion currents to determine
peptide abundance
Protein Profiling
• Generate large scale proteome maps
• Annotate and correct genomic sequences
• Analyze protein expression as a function of
cellular state
Analysis of Plasmodium falciparum
(malaria parasite) proteome
Figure 1: Proteins identified in each stage are plotted as a function of their broad
functional classification. To avoid redundancy, only one class was assigned per
protein.
Florens, L. et al. (2002) A proteomic view of the Plasmodium falciparum life cycle.
Nature. 419, 520-526.
Analysis of Myc oncogene proteome
Fig. 3. Summary of functionally
related expression changes in
Myc(+) cells. The proteins
reduced or induced in Myc(+)
cells are shown in green or red,
respectively. The numbers
denote fold expression change.
The arrows denote activation
and the blocked lines denote
inhibition.
Shiio, Y. et al. (2002) Quantitative
proteomic analysis of Myc oncoprotein
function. EMBO J. 21, 5088–5096.
Protein Interactions
• When analyzing a new protein, first question
to ask is – to what proteins does it bind?
• Method: Use new protein as an affinity agent
to isolate its binding partners
– Will not detect low affinity, transient, or cellular
environment specific interactions
Protein Interaction Experiments
• Steps
1. Bait presentation
using endogenous
proteins
2. Affinity purification
of complex
3. Analysis of bound
proteins
Studies of Large Protein Complexes
• Spliceosome in yeast and human cells
• Nuclear pore complex in yeast
• Nucleolus in human cells
– Largest organelle mapped
– Found over 400 nucleolar proteins
– Still not complete
Analyzing Protein Modifications
• Finding all modifications on a single protein
– Identified by examining the measured mass and
fragmentation spectra
• Proteome wide scanning of modifications
– Not complete
Additional Challenges
• Experimental design
– Large amounts of data, absence of hypotheses
– Must take advantage of statistical methods
• Data collection
– High throughput collection
– High quality data
• Data analysis, visualization, and storage
• Data Publication
Future Directions
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Influence on clinical diagnostics and therapy
Analysis of whole proteins
Tissue imaging
Using mass tags for high throughput protein
identification
Other Applications of
Mass Spectrometry
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Isotope dating and tracking
Trace gas analysis
Mapping the location of individual atoms
Pharmacokinetics
Space exploration
Respiratory gas analysis
Conclusion
Proteomics is extremely valuable for
understanding biological processes and
advancing the field of systems biology.
“The ultimate goal of systems biology is the
integration of data from these observations
into models that might, eventually, represent
and simulate the physiology of the cell.”
Proteomics Websites
Uniprot http://www.uniprot.org/
Interpro http://www.ebi.ac.uk/interpro/