Lecture__on__Proteomics_Introduction.ppt
- 1. DNA Genome “Genomics” Proteins Cell functions Proteome “Proteomics” DNA sequencing cDNA arrays 2D PAGE, HPLC CGTCCAA CTGACGT CTACAAG TTCCTAA GCT RNA Transcriptome “-ome” Reactome, the chemical reactions involving a nucleotide
- 2. Protein Chemistry/Proteomics Protein Chemistry • Individual proteins • Complete sequence analysis • Emphasis on structure and function • Structural biology Proteomics • Complex mixtures • Partial sequence analysis • Emphasis in identification by database matching • System biology
- 3. Proteins are the mediators of functions in the cell Deviations from normal status denotes disease Proteins are drug/therapeutic targets Why are we studying proteins?
- 4. Proteome Mining Identifying as many as possible of the proteins in your sample Protein Expression Profiling Identification of proteins in a particular sample as a function of a particular state of the organism or cell Functional proteomics Post-translational modifications Identifying how and where the proteins are modified Protein-protein interactions Protein- network mapping Determining how the proteins interact with each other in living systems Structural Proteomics Protein quantitation or differential analysis Proteomics and biology /Applications
- 5. Tools of Proteomics Protein separation technology Simplify complex protein mixtures Target specific proteins for analysis Mass spectrometry (MS) Provide accurate molecular mass measurements of intact proteins and peptides Database Protein, EST, and complete genome sequence databases Software collection Match the MS data with specific protein sequences in databases
- 6. The Proteome • Cycle of Proteins • Proteins as Modular Structures – motifs, domains • Functional Families • Genomic Sequences • Protein Expression /Protein level The proteome in any cell represents a subset of all possible gene products Not all the genes are expressed in all the cells. It will vary in different cells and tissue types in the same organism and between different growth and developmental stages The proteome is dependent on environmental factors, disease, drugs, stress, growth conditions.
- 7. Life cycle of a protein Information found in DNA is used for synthesis of the proteins mRNA Protein Proteolytic Cleaveage Acylation Methylation Phosphorylation Sulfation Selenoproteins Ubiquination Glycolisation Translocation Damage -free radicals Degradation Environmental -chemicals radioactiivty Posttranslational Processing to specific subcellular or extracellular compartments Folding
- 8. Molecular Structures -helices -sheets Primary structure a chain of amino acids Secondary structure three dimensional form, formally defined by the hydrogen bonds of the polymer Amino acids vary in their ability to form the various secondary structure elements. Confer similar properties or functions when they occur in a variety of proteins Amino acids that prefer to adopt helical conformations in proteins include methionine, alanine, leucine, glutamate and lysine ("MALEK" in amino acid 1-letter codes) The large aromatic residues (tryptophan, tyrosine and phenylalanine) and Cβ-branched amino acids (isoleucine, valine and threonine) prefer to adopt -strand conformations.
- 9. Sequence alignment A software tool used for general sequences alignment tasks is ClustalW The degree of relatedness, similarity between the sequences is predicted computationally or statistically Sequence alignment is a way of arranging primary sequences (of DNA, RNA, or proteins) in such a way as to align areas sharing common properties.
- 10. ClustalW
- 11. BLAST It is used to compare a novel sequence with those contained in nucleotide and protein data bases by aligning the novel sequence with the previously characterized genes. The emphasis of this tools is to find regions of sequence similarity, which will yield functional and evolutionary clues about the structure and function of this novel sequence. Basic Local Alignment Search Tool NCBI BLAST http://www.ncbi.nlm.nih.gov/BLAST/
- 13. Molecular Structures / Functional Families Tertiary structure the overall shape of the protein (fold) the process by which a protein assumes its characteristic function The three-dimensional shape of the proteins might be critical to their function. For example, specific binding sites for substrates on enzymes Specific sequences that also confer unique properties and functions, motifs or domains Quaternary structure -formation usually involves the "assembly" or "coassembly" of subunits that have already folded Incorrectly folded proteins are responsible for illnesses such as Creutfeltdt_Jakob disease and Bovine spongiform encephalopathy (mad cow disease), and amyloid related illnesses such as Alzheimer’s.
- 14. Domains / Motifs Structural alignment: a method for discovering significant structural motifs. -based on comparison of shape Structural alignment of thioredoxins from humans (red)and the fly Drosphila melangaster (yellow). Motifs: short conserved sequences, which appear in a variety of other molecules. Domains: part of the sequence that appear as conserved modules in proteins that are not related, in global terms. Usually with a distinct three dimensional fold, carrying a unique function and appearing in different proteins Repeats: structurally or functionally interdependent modules.
- 15. Functional families Protein family classification databases: PROSITE. Database of protein families and domain, defined by patterns and profiles, at ExPASY. http://au.expasy.org/prosite/ Pfam. Multiple sequence alignments and HMMs of protein domains and families, at Sanger Institute. http://www.sanger.ac.uk/Software/Pfam/help/index.shtml SMART Simple Modular Architecture Research Tool, at EMBL. http://smart.embl-heidelberg.de/ By associating a novel protein with a protein family, one can predict the function of the novel protein Domains are clustered into families in which significant sequence similarity is detected as well as conservation of biochemical activity. SCOP-a structural classification of proteins Proteins can be grouped into functional families; proteins that carry out related functions Structural Signaling pathways Metabolic Transportation
- 17. A Pseudo-Rotational Online Service and Interactive Tool
- 18. Pfam
- 20. Sequence Threading Sequence-Structure-Function Structure more conserved than sequence Structure Function Threading techniques try to match a target sequence on a library of known three-dimensional structures by “threading” the target sequence over the known coordinates. In this manner, threading tries to predict the three-dimensional structure starting from a given protein sequence. It is sometimes successful when comparisons based on sequences or sequence profiles alone fail to a too low similarity. (modified from: http://www.pasteur.fr/recherche/unites/Binfs/definition/bioinformatics_definition.html)
- 21. Genomic sequencing/ Protein level Genome size (bp) 5.386 580.000 12,1 106 3,2 109 90 109 670 109 Mycoplasma genitalium Yeast (S. Cerevisiae) X-174 virus Human Lilium longiflorum Amoeba dubia Biological complexity does not come simply from greater number of genes. complexity
- 22. Complexity
- 24. More than 100 modification forms known A single protein may carry several modifications Modified proteins show different properties compared to unmodified counterparts In most cases, we do not know the origin or the biological significance of the observed heterogeneities Much larger number of spots compared to protein species they represent H.influenza : 1500 spots 500 different proteins Protein Heterogeneity
- 25. 4.5 pI Electrophoresis, 1999, 20 (14) 2970 g-enolase About 3000 Spots after Coomassie Stain Partial 2D-gel images showing g-enolase from human brain. The protein is represented by one spot when IEF was performed on pH 3-10 non-linear IPG strips (A), and by six spots when IEF was performed on pH 4-7 strips (B). Increased Resolution and Detection of More Spots with the Use of Narrow pH Gradient Strips A B 2D gel image of brain proteins
- 27. Genomic sequencing Paralogues are similar sequences within a single organism that have arisen due to a gene duplication event. Homologues are similar sequences in two different organisms that have been derived from a common ancestor sequence. Orthologues are similar sequences in two different organisms that have arisen due to a speciation event.
- 28. Pattern / Profile Pattern –conserved sequence of a few amino acids identify various important sites within protein •Enzyme catalytic site •Prosthetic group attachment •Metal ion binding site •Cysteines for disulphide bonds •Protein or molecular binding Profile a multiple alignment with matrix frequencies- describe protein families or domains conserved in sequence. •Score-based representations •Position-specific scoring matrix (PSSM) •Hidden Markov model (HMM) Database: PROSITE Patterns Patterns and Profiles aredused to search for motifs/ domains of biological significance that characterize protein family
- 29. Protein level Codon bias- the tendency of an organism to prefer certain codons over others that code for the same amino acid in the gene sequence. The level of any protein in a cell at a given time: • Transcription rate • Efficiency of translation in the cell • The rate of degradation of the protein Larger genomes have larger gene families (the average family size also increases with genome size)
- 30. Protein expression Protein It consists of the stages after DNA has been translated Amino acid chains chains which is ultimately folded into proteins Expression profiling what genes are expressed in a particular cell type of an organism, at a particular time, under particular conditions? As the expression of many genes is known to be regulated after transcription, an increase in mRNA concentration need not always increase expression
- 31. ESI-MS Electrospray Ionization tandem MS MALDI-TOF Matrix Assisted Laser Desorption Ionization –Time of Flight General workflow of proteomics analysis proteins digestion separation MALDI, MS/MS digestion peptides (LC)-MS/MS Identification
- 32. The less complex a mixture of proteins is, the better chance we have to identify more proteins. Detergents Reductants Denaturing agents Enzymes digestion Separation of Protein Mixtures
- 33. Separation techniques 1D- and 2D-SDS PAGE Preparative IEF isoelectric focusing HPLC Separating intact proteins to take advantage of their diversity in physical properties Separation techniques for peptides MS-MS HPLC (MudPIT) Multi-Dimensional Protein Identification Technology SELDI Surface-enhanced laser desorption/ionization Separation techniques used with intact proteins Differential display proteomics Difference gel electrophoresis (DIGE) Isotope-coded affinity tagging (ICAT)
- 34. •Enrichment from larger volumes Selective precipitation Selective centrifugation Preparative approaches •Combination of 2DE with LC •Multi-dimensional LC Enrichment /Fractionation For the detection of low-abundance proteins, a separation of complex mixtures into fractions with fewer components is necessary
- 35. Detergents: solubilize membrane proteins- separation from lipids Reductants: Reduce S-S bonds Denaturing agents: Disrupt protein-protein interactions-unfold proteins Enzymes: Digest contaminating molecules (nucleic acids etc) Protease inhibitors Aim: High recovery-low contamination-compatibility with separation method Protein extraction
- 36. Why digest the protein? Accuracy of mass measurements Suitability Sensitivity Good activity both in gel digestion and in solution The ideal protein digestion approach would cleave proteins at certain specific amino acid residues to yield fragments that are most compatible with MS analysis. Peptide fragments of between 6 – 20 amino acids are ideal for MS analysis and database comparisons. Other enzymes with more or less specific cleavage: Chymotrypsin Glu C (V8 protease) Lys C Asp N Protein digestion Trypsin Cleaves at lysine and arginine, unless either is followed by proline in C- terminal direction
- 37. Gel electrophoresis Classical process High resolving power: visualization of thousands of protein forms Quantative Identifying proteins within proteome Up/ down regulation of proteins Detection of post-translational modifications Silver: www.healthsystem.virginia.edu Ruby: www.komabiotech.co.kr Protein fixing and staining or blotting General detection methods (staining) Organic dye – and silver based methods Coomassie blue, Silver Radioactive labeling methods Reverse stain methods Fluorescence methods (Supro Ruby) Gel scanning (storage of image in a database) Coomassie blue stained gels Silver stained Ruby red
- 38. Isoelectric point •Proteins are amphoteric molecules i.e. they have both acidic and basic functional groups •pI= isoelectric point, is where the protein does not have any net charge •The protein charge depends on the pH of the solution.
- 39. A pH gradient is generated by a limited number of well defined chemicals (immobilines) which are co-polymerized with the acrylamide matrix. Migration of proteins in a pH gradient: protein stop at pH=pI Loading quantities (18 cm strip) Analytical run: 50-100 μg Micropreparative runs: 0,5 – 10 mg Individual strips: 24,18,11,7 cm long 3 mm wide 0,5 mm thickness Use narrow range IPG strips to focus on particular pI range Immobilized pH gradients (IPGs) 1st dimension IsoElectric Focusing, IEF
- 40. 2nd dimension pH 3 pH 10 The strip is loaded onto a SDS gel Mw pI Staining ! Proteins that were separated on IEF gel are next separated in the second dimension based on their molecular weights.
- 41. Limitations/difficulties with the 2D gel Reproducibility Samples must be run at least in triplicate to rule out effects from gel-to-gel variation (statistics) Incompatibility of some proteins with the first dimension IEF step (hydrophobic proteins) Marginal solubility leads to protein precipitation and degradation- smearing (Glycolysation, oxidation) Small dynamic range of protein staining as a detection technique- visualization of abundant proteins while less abundant might be missed. Posttranscriptional control mechanisms Co-migrating spots forming a complex region Streaking and smearing Weak spots and background
- 42. 4.5 9.5 pI 90 20 kDa A B Brain Proteins (About 3000 Spots after Coomassie Stain) Electrophoresis, 1999, 20 (14) 2970
- 43. A B g-enolase Partial 2D-gel images showing g-enolase from human brain. The protein is represented by one spot when IEF was performed on pH 3-10 non-linear IPG strips (A), and by six spots when IEF was performed on pH 4-7 strips (B). Protein Heterogeneity Increased Resolution and Detection of More Spots with the Use of Narrow pH Gradient Strips
- 44. Vacuum assisted aspiration into sample tubes Large amount of proteins (up to 3g protein) Preparative IEF The protein mixture is injected into the focusing chamber Proteins are focused as in standard IEF The pH gradient is achieved with soluble ampholytes
- 45. DIGE Proteins are labeled prior to running the first dimension with up to three different fluorescent cyanide dyes Allows use of an internal standard in each gel-to-gel variation, reduces the number of gels to be run Adds 500 Da to the protein labeled Additional postelectrophoretic staining needed Quantification of Spot Relative Levels 2D Fluorescence Difference Gel Electrophoresis
- 46. Separation by LC modified:www.dcu.ie/chemistry/ssg/image s/Techni7.gif Salt gradient UV detector EC detector column waste Number of peaks indicates the complexity of starting material Peak position (i.e. elution time) may provide qualitative information about the sample (comparison with standards) Peak area may provide information on relative concentration of components. If coupled to MS protein identification (MW) can be provided
- 47. Multidimensional HPLC Mud PIT Multidimensional Protein Identification Techniques or Tandem HPLC the combination of dissimilar separation modes will allow a greater resolution of peptides in mixture. Ion-exchange Reversed phase •Reversed phase, hydrophobicity •Ion exchange, net positive/negative charge •Size exclusion, peptide size, molecular weight •Affinity chromatography, interaction with specific functional groups
- 49. The sample has to be introduced into the ionization source of the instrument. Once inside the ionization source the sample molecules are ionized, because ions are easier to manipulate than neutral molecules. These ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass (m)-to-charge (z) ratios (m/z). The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum. The analyzer and detector of the mass spectrometer, and often the ionization source too, are maintained under high vacuum to give the ions a reasonable chance of traveling from one end of the instrument to the other without any hindrance from air molecules. Modified from www.csupomona.edu/~drlivesay/ Chm561/winter04_561_lect1.ppt A Mass Spectrometer source analyzer detector
- 50. ..consists of.. Detector –detection of mass separated ions source analyzer detector MALDI, Matrix-Assisted Laser Desorption and Ionisation ESI, ElectroSpray Ionisation Source -produces the ions from the sample (vaporization /ionization) Mass Anlyzer - resolves ions based on their mass/charge (m/z) ratio Generate different, but complementary information
- 51. MALDI Matrix Assisted Laser Desorption and Ionisation Peptides co-crystallised with matrix Produces singly charged protonated molecular ions High throughput Single proteins Rapid procedure, high rate of sample throughput large scale identification (“first look at a sample”) + + - + - + laser ions + - - +
- 52. TOF Separate ions o f different m/z based on flight time Fast Requires pulsed ionization Time of flight Measures the time it takes for the ions to fly form one end to other and strike the detector. The speed with which the ions fly down the analyzer tube is proportional to their m/z values. The greater the m/z the faster they fly
- 53. Matrix-assisted laser desorption ionization-time of flight MALDI-TOF + + - + - + + - - + Quick, easy, inexpensive Highly tolerant to contaminents High sensitivity Good accuracy in mass determination Compatible with robotic devices for high-throughput proteomics work Best suited to measuring peptide masses TOF analyzer Low reproducibility and repeatability of single shot spectra (Averaging) Low resolution Matrix ions interfere in the low max range
- 54. MALDI-TOF data Peak List = List of masses 112.1 234.4 890.5 1296.9 1876.4 1987.5 ……. = Modified from http://plantsci.arabidopsis.info/pg/day3practical1.ppt Every peak corresponds to the exact mass (m/z) of a peptide ion fingerprint
- 55. ElectroSpray Ionization, ESI Voltage Ions are generated by spraying a sample solution through a charged inlet Produces multiply protonated molecular ions of biopolymers ++ + ++ + + + ++ Capillary column Charged droplets + + + + Peptide ions Heated desolvation region •Nanospray needles, fine tipped gold coated needles •Single samples •Nanospray LC probe, connects directly to HPLC outlet – automated sample injection •Samples in solution •Compatible with HPLC •Complex mixtures •Tandem MS analysis •Peptide sequence
- 56. Analyzers Detector –detection of mass separated ions source analyzer detector MALDI, Matrix-Assisted Laser Desorption and Ionisation ESI, ElectroSpray Ionisation Source -produces the ions from the sample (vaporization /ionization) Mass Anlyzer - resolves ions based on their mass/charge (m/z) ratio Time of Flight, TOF The Quadrupole, Q Ion Trap
- 57. The Quadrupole The quadrupole consists of four parallel metal rods. Ions travel down the quadropole in between the rods. Only ions of a certain m/q will reach the detector for a given ratio of voltages: other ions have unstable trajectories and will collide with the rods. This allows selection of a particular ion, or scanning by varying the voltages. source Voltage Filters out all m/z values except the ones it is set to pass Obtains a mass spectrum by sweeping across the entire mass range
- 58. Collects and store ions in order to perform MS-MS analyses on them. Ion Trap Mass Analyzer Trapped ions Ions in Ions out The trap consists of a top and a bottom electrode and a ring electrode around the middle. Ions are ejected on the basis of their m/z values. To monitor the ions coming from the source, the trap continuoulsy repeats a cylcle of filling the trap with ions and scanning the ions according to their m/z values. Separates the mass analysis and ion isolation events in time (using a single mass analyzer) Ionization ion transfer/trapping parent ion isolation/ fragmentation daughter ion detection
- 59. A mass analyzer for determining the mass-to-charge ratio (m/z) of ions based on the cyclotron frequency of the ions in a fixed magnetic field. All ions are detectedall ions are detected simultaneously over some given period of time Ions are injected into a magnetic field , that causes them to travel in circular paths. Excitation with oscillating electrical field increases the radius and enables a frequency measurement Fourier Transform MS Fourier transform ion cyclotron resonance mass spectrometry, FTICMS ICR can be used with different ionization methods, ESI, MALDI A short sweep of frequencies is used to excite all ions. The complex spectrum of intensity/time is analyzed with Fourier Transform to extract the m/z componets High resolution High accuracy Very sensitive (the minimal quantity for detection is in order of several hundered ions Non destructive –the ions don’t hit the detection plate so they can be selected for further fragmentation
- 60. Sensitivity amounts of proteins are limited Resolution how well we can distinguish ion of very similar m/z values (the ability of the instrument to resolve two closely placed peaks in the mass spectrum) Mass accuracy the measured values for the peptide ions must be as close as possible to their real values. (the relative percent difference between the measured mass and the true mass, usually represented in ppm.) type m/z range Resolving power cost Quadrupole 1-4000 1000 $$ Ion trap 10-4000 1000 $$ Time of flight 1-100.000 30.000 $$$ Fourier transform 18-10.000 >100.000 $$$$ Figures of merit for mass analyzers MS
- 61. R = m/Δm = m/(m2-m1) Mass Resolution intensity The ability of the instrument to resolve two closely placed peaks.
- 62. Mass accuracy The relative percent difference between the measured mass and the true mass (usually represented in ppm). (The lower the number the better the mass accuracy)
- 63. Molecular ion / precursor ion Ion formed by ionization of the analyte species Fragment ions / product ions Ions formed by the gas-phase dissociation of the molecular ion Relative Abundance Relative Abundance is a measure of the relative amount of ion signal recorded by the detector MS/MS terminology
- 64. Hybrid instruments /Tandem MS Combines two or more mass analyzers of the same or different types First mass analyzer isolates the ion of interest (parent ion) The ions are then fragmented between the first and second mass analyzer via collisions or irridation with UV light The last mass analyzer obtains the mass spectrum of the fragments ions (daughter ions spectrum) MS-MS spectra reveal fragmentation patterns to provide structural information about a molecule Protein identification by cross-correlation algorithms
- 65. The triple Quadrupole Mass analyzer Mass analyzer Detector Mixture Survey scan Mass analyzer Mass analyzer Detector Mixture Isolated species Fragments MS/MS scan Collision cell Modified fromÖ Christophe D. Masselon, CEA Grenoble Full-scan, rapid scanning of Q1, values of all ions coming from the source at any given moment are recorded The first quad (Q1) will act as a mass filter in which the voltage settings are fixed to allow only ions of a specific m/z value to pass through. The peptide ions then enter Q2, where they collide with argon gas, to fragment the parent ion present (collision induced dissociation, CID) The third quad (Q3) scans repeatedly over a mass range to detect the fragment ions, obtaining a spectrum.
- 66. Q-TOF Quadruple Time of Flight mass analyzer Higher mass resolution, increased mass accuracies More effectively used in software- assisted data interpretation
- 67. SELDI Advantages of SELDI technology: Uses small amounts (< 1l/ 500-1000 cells) of sample (biopsies, microdissected tissue). Quickly obtain protein mapping from multiple samples at same conditions. Ideal for discovering biomarkers quickly. Surface Enhanced Laser Desorption Ionization A combination of chromatography (protein chips) and MALDI-TOF MS Protein capture and enrichment on a chemically or bio affinity active solid phase surface washing EAM, energy absorbing molecule Retained proteins are “eluted” from the Protein Chip array by Laser Desorption and Ionization Ionized proteins are detected and their mass accurately determined by Time-of- Flight Mass Spectrometry
- 68. Chemical Surfaces (Hydrophobic) (Anionic) (Metal Ion) (Normal Phase) (Cationic) (Antibody - Antigen) (Receptor - Ligand) (DNA - Protein) (PS10 or PS20) Biological Surfaces The chip
- 69. Software for MS PeptIdent MultiIdent ProFound PepSea MASCOT MS-Fit SEQUEST PepFrag MS-Tag Sherpa Task for students: find the appropriate url for each above mentioned tool