Mass spectrometry and proteomics Eva Dimitrova and Jessica Connor

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

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

Step 1: Sample prep Step 2: Separation Step 3: Mass spectrometry 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 - 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: ~1500-2500 proteins

Staining Technology Staining Sypro Ruby-$$$ Silver Coomassie blue Fluorescent dyes Sypro Ruby-$$$ 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 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.

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 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

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 Spot removed from gel Fragmented using trypsin Spectrum of fragments generated MATCH Library Artificial spectra built 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 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…

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 Any other advantages or uses?

The Human Proteome Initiative. (2007) http://ca. expasy 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 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

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, 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. 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.

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 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

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 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

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

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

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/ 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

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