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Mass spectrometry and proteomics

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Isotope dating and tracking. Trace gas analysis. Mapping the location of individual atoms ... Proteomics Websites. Uniprot http://www.uniprot.org ... – PowerPoint PPT presentation

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Title: Mass spectrometry and proteomics


1
Mass spectrometry and proteomics
Mass spectrometry and proteomics
Eva Dimitrova and Jessica Connor
2
The omics nomenclature
3
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

4
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

5
Components of Expressional Proteomics
Protein Separation
Mass Spectroscopy
Bioinformatics
6
Pathway
  • Step 1 Sample prep
  • Step 2 Separation
  • Step 3 Mass spectrometry

7
Movie
8
General overview
Aebersold, R Mann, M. (2003, March). Mass
spectrometry based proteomics. Nature. 422,
198-207
9
  • Sample preparation

10
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

11
Cleanup and fractionation
  • General Purpose Cleanup
  • Improve Resolution
  • Improve Reproducibility
  • Fractionation
  • Reduce Complexity
  • Improve Range of Detection
  • Enrich low-abundance proteins

www.expressionproteomics.com
12
  • Separation

13
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
14
Staining Technology
  • Staining
  • Silver
  • Coomassie blue
  • Fluorescent dyes
  • Sypro Ruby-
  • Radioisotopic labeling

15
Trypsin digestion
  • Trypsin
  • Serine protease
  • Claves at the carboxyl end of lysine and arginine
    (except when either is followed by proline)

16
Mass Spectrometry
17
How does a mass spectrometer work?
Create ions
Separate ions
Detect ions
  • Mass spectrum
  • Database analysis
  • Ionization method
  • MALDI
  • Electrospray
  • (Proteins must be charged and dry)
  • Mass analyzer
  • MALDI-TOF
  • Quadrapole
  • MALDI-QqTOF
  • AA seq and MW
  • QqTOF
  • AA seq and protein modif.

18
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.

19
Mass analyser
  • TOF time of flight
  • Ion trap
  • Quadropole
  • Fourier transform ion cyclotron

20
Mass Spec Principles
Sample

_
Detector
Ionizer
Mass Analyzer
21
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
22
ESI and MALDI
Aebersold, R Mann, M. (2003, March). Mass
spectrometry based proteomics. Nature. 422,
198-207
23
Peptide sample
24
Stable isotope protein labeling
Aebersold, R Mann, M. (2003, March). Mass
spectrometry based proteomics. Nature. 422,
198-207
25
Peptide Mass Identification
Spectrum of fragments generated
MATCH
Library
Database of sequences (i.e. SwissProt)
26
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
    di-peptides, tri-peptides, tetra-peptides

Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc
27
Peptide sample
28
Peptide Hits
29
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
30
Proteomics Applications
31
Why Proteomics?
  • Proteins are the active biological agents in
    cells
  • DNA sequences dont show how proteins function or
    how biological processes occur
  • Proteins undergo post transcriptional
    modifications
  • 3D structures affect protein function
  • Alternative splicing

32
The Human Proteome Initiative. (2007)
http//ca.expasy.org/sprot/hpi/hpi_desc.html
Retrieved March 24, 2009.
33
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

34
Protein Profiling
  • Generate large scale proteome maps
  • Annotate and correct genomic sequences
  • Analyze protein expression as a function of
    cellular state

35
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.
36
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,
50885096.
37
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

38
Protein Interaction Experiments
  • Steps
  • 1. Bait presentation
  • using endogenous
  • proteins
  • 2. Affinity purification
  • of complex
  • 3. Analysis of bound
  • proteins

39
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

40
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

41
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

42
Future Directions
  • Influence on clinical diagnostics and therapy
  • Analysis of whole proteins
  • Tissue imaging
  • Using mass tags for high throughput protein
    identification

43
Other Applications of Mass Spectrometry
  • Isotope dating and tracking
  • Trace gas analysis
  • Mapping the location of individual atoms
  • Pharmacokinetics
  • Space exploration
  • Respiratory gas analysis

44
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.

45
Proteomics Websites
46
Uniprot http//www.uniprot.org/
47
Interpro http//www.ebi.ac.uk/interpro/
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