1. Mass spectrometry and proteomics
Eva Dimitrova and Jessica Connor

2. The “omics” nomenclature…
Genomics
DNA (Gene)
Functional
Transcriptomics
RNA
Proteomics
PROTEIN
Metabolomics
METABOLITE
Transcription
Translation
Enzymatic
reaction

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.”

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. Step 1: Sample prep Step 2: Separation Step 3: Mass spectrometry
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,

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

12. Separation

13. 2D-SDS PAGE gel - gel with an immobilised pH gradient
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: ~ proteins

14. Staining Technology Staining Sypro Ruby-$$$ Silver Coomassie blue
Fluorescent dyes
Sypro Ruby-$$$
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 + _
Ionizer
Mass Analyzer
Detector

21. Typical Mass Spectrum m/z ratio: Molecular weight
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,

23. Peptide sample

24. Stable isotope protein labeling
Aebersold, R & Mann, M. (2003, March). Mass spectrometry based proteomics. Nature. 422,

25. Peptide Mass Identification
Spot removed from gel
Fragmented using trypsin
Spectrum of fragments generated
MATCH
Library
Artificial spectra built
Artificially trypsinated
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 don’t show how proteins function or how biological processes occur
Proteins undergo post transcriptional modifications
3D structures affect protein function
Alternative splicing
Any other advantages or uses?

32. The Human Proteome Initiative. (2007) http://ca. expasy
The Human Proteome Initiative. (2007) 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
Large Scale Proteome Maps
Deinococcus radiodurans genome 60% complete
Plasma Proteome Project – run by Human Proteome Organization
The long term goals of this project are:
Comprehensive analysis of plasma and serum protein constituents in people
Identification of biological sources of variation within individuals over time, with validation of biomarkers
Physiological: age, sex/menstrual cycle, exercise
Pathological: selected diseases/special cohorts
Pharmacological: common medications
Determination of the extent of variation across populations and within populations
Challenges with human plasma analysis
Expected to contain thousands, possibly millions of proteins
Only 500 have been reported
Annotating and Correcting Genome Sequences
Found previously undetected genes in yeast and human genomes
Can differentiate between alternatively spliced or translated forms of a protein
Analysis of protein expression as a function of cellular state
Most common use of proteomics
Ties into understanding the function of proteins
Add to gene ontology

35. Analysis of Plasmodium falciparum (malaria parasite) proteome
Found over 200 candidate proteins for stage specific drug or vaccine targets
Stage specificity of surface proteins in mosquito and human stages
Study published in Nature
Used tandem MS and other methods to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes). Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Found a potential mechanism for controlling gene expression.
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,

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.
Used tandem MS
The global pattern of protein expression in rat myc-null cells was compared with that of myc-plus cells to generate a differential protein expression catalog.
Identified two new functions (reduction of stress fibres and focal adhesions)
Shiio, Y. et al. (2002) Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21, 5088–5096.

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
General Purification Method: Isolate protein of interest by tagging it with a sequence readily recognized by an antibody specific for the tag
Works well in yeast
Tagged proteins in mammalian cells prone to artefacts
Tandem Affinity Purification (TAP) can be used to remedy this. This method will be discussed more in a presentation later in the semester
The tandem-affinity-purification (TAP) tag consists of three components: a calmodulin-binding peptide, a tobacco etch virus (TEV) protease cleavage site and Protein A as an immunoglobulin G (IgG)-binding domain. Cells or organisms are generated that contain TAP-tagged protein(s). Extracts are then prepared under mild conditions and TAP is carried out. The first column consists of IgG beads. TEV protease cleaves the immobilized multiprotein complexes. Another round of binding is carried out on a second column that consists of calmodulin beads. The native complex is then eluted by chelating calcium using EGTA.
Pro: reduce background noise
Con: lose transient and weak binding interactions

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
Large amounts of data with absence of hypotheses but you still need an experiment that will have accurate and organized data that can formulate some sort of outcome
In order to take advantage of statistical methods you need
Controlled repeat studies
Generation of models describing the source, magnitude and distribution of errors
Data analysis, visualization, and storage
Can’t manually analyze everything
Need to develop tools for analyzing proteomic data
Need to develop transparent file structures for data storage, communication and visualization
Data Publication
Need to find ways to review and validate large data sets
Need to make information electronically searchable
These challenges are still for the most part unresolved

42. Future Directions Influence on clinical diagnostics and therapy
Analysis of whole proteins
Tissue imaging
Using mass tags for high throughput protein identification
Clinical Diagnostics
Cancer diagnosis
serum protein patterns that distinguished patients with ovarian cancer from unaffected women with a positive predictive value of 94%
One of the first biomarkers used in disease diagnosis was prostate-specific antigen (PSA). Today, serum PSA levels are commonly used in diagnosing prostate cancer in men.
NCI reports three candidate proteins in the blood that distinguish people with breast cancer
Human Proteome Project at the UWSMPH
Mission is to use proteomics to develop disease treatment and detection methods
Human Proteome Initiative
Annotate all human proteins and mammalian orthologs of human proteins
Annotation of all known human polymorphisms at the protein sequence level
Annotation of all known post-translational modifications in human proteins
Linking findings to structural information

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
Pharmacokinetics – determining the fate of drugs administered to an organism
Space exploration – mass spectrometers taken to Mars and Saturn’s largest moon to analyze atmospheric samples
Used to analyze respiratory gas in hospitals in the past

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/
UniProt is the universal protein resource, a central repository of protein data created by combining Swiss-Prot, TrEMBL and PIR. This makes it the world's most comprehensive resource on protein information.
Very helpful site tour

47. Interpro http://www.ebi.ac.uk/interpro/