Transcript Document
Proteomics Dimitri Raptis & Alexander Kögel Protein identification using mass spectrometry www.draptis.eu/proteomics.ppt Introduction Proteomics important technology Large-scale study of proteins Structure Functions “Omics” revolution: shift in strategy piece-by-piece -> global analysis hypothesis-driven -> discovery-based research Expression proteomics analysis of differential protein expression Functional proteomics posttranslational modifications protein-protein, protein-ligand interactions sequence-structure-function relationships Twyman RM (2004). Principles Of Proteomics. Oxford, UK: BIOS Proteins Biochemical compounds 1 > polypeptides Single linear polymer chain of amino acids (AA) Bonded together by peptide ponds – carboxyl & AA residues ”Proteins." Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Amino acid sequence ”Proteins." Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Protein structure Weaver, Molecular Biology, McGraw Hill: Boston, 2002 Proteome Entire set of proteins expressed by Genome Cell Tissue Organism > 400.000 proteins, dynamic Human proteome Aebersold et al, Systems biology - ETH Zurich, https://www.e-pics.ethz.ch/ Why proteomics? Why proteomics? Protein alterations cannot be fully deduced from DNA. RNA expression does not always reflect protein levels: translational control degradation turnover Some tissues not suitable for RNA expression analysis. Proteins are the physiological/pathological active key players. General goal: better understanding of genesis and progression of diseases Clinical goals: early disease detection (biomarkers) identification of therapeutic targets therapy monitoring Multidisciplinary Cells, tissue HPLC MALDI, ESI TOF, Q, IT Algorithms Typical stages Aebersold R, Mann M, Nature. 2003 Internal standards Tandem mass tags Isotope dilution Quantification strategies Domon B, Aebersold R. Science. 2006 Top down or bottom up? Bottom-up Starting with proteolytic fragments Piecing the protein back together de novo repeat detection Fragment ions of peptides MS/MS Proteolytic digest e.g. Trypsin Bottom-up Most common Protein Tandem MS of whole protein ions Pulling them apart Electron capture dissociation MS/MS Fragment ions of protein Top down Top down Extensive sequence information ”Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Separation Specific protein biophysical parameters Isoelectric point Molecular weight Affinity Chromatographic methods HPLC 2D-HPLC ProteinChips Electrophoretic methods SDS-PAGE 2-D E Reverse phase (RPLC) – Hydrophobicity Yates JR, et al. Annu Rev Biomed Eng. 2009 2D Gel electrophoresis 1D: isoelectric focussing (IEF) separation by IP 2D: dimension: SDS-PAGE separation by MW staining > 1000 proteins /gel molecular analysis by MS HPLC Westernblot Pitfalls very basic / acidic; large / small; hydrophobic; low-abundance proteins Fontana et al. Proteomics 2004 Robotic isolation ”Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Shotgun proteomics HPLC ”HPLC" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. HPLC HPLC/MS Peptides from protein mixture fractionated in steps Eluent ESI-MS MALDI: series ”HPLC" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Protein & peptide fractionation Complex mixtures Proteins Molecules Works only if mixture has equal amounts Abundant species suppress signals from less abundant ones Difficult to interpret Enyzmatic digestion -> many peptide products 2-D electrophoresis Protein fractionation High performance liquid chromatography Peptide fractionation ”Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Proteins Peptides Fragments Proteases, e.g. trypsin, break protein into peptides Tandem MS breaks peptides into fragment ions Measures the mass of each piece. MS accelerates the fragmented ions; heavier ions accelerate slower than lighter ones MS measure mass/charge ratio of an ion Collision Induced Dissociation H+ H...-HN-CH-CO Ri-1 Prefix Fragment . . . NH-CH-CO-NH-CH-CO-…OH Ri Ri+1 Suffix Fragment Peptides tend to fragment along the backbone. Fragments can also loose neutral chemical groups like NH 3 and H2O. Ionisation Proteins Polar Thermally unstable Ionisation transfers analyte into gas phase No degradation Matrix-assisted laser desorption ionization (MALDI) Laser nitrogen beam (soft) Matrix protection (Sinapinic acid) Electrospray ionization (ESI) No fragmentation MALDI Nonvolatile Mass spectrometry WE Stephens 1952 Patent Mass spectrometry Analytical technique mass-charge ratio (m/z) charged particles Ion source Mass analyzer Detector ”Mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. Mass analysers Scanning and ion-beam mass spectrometers TOF and Q Trapping mass spectrometers IT and Orbitrap Whole protein mass analysis time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR). Mass analysis of proteolytic peptides more popular Low costs Sample preparation is MALDI time-of-flight instruments Multiple stage quadrupole-time-of-flight and quadrupole ion trap also find use in this application. Mass spectrometers Base peak chromatogram Yates JR, et al. Annu Rev Biomed Eng. 2009 Mass chromatogram Yates JR, et al. Annu Rev Biomed Eng. 2009 Label-free analysis Isotopic labeling Quantitative analysis Yates JR, et al. Annu Rev Biomed Eng. 2009 Quantitative proteomics Yates JR, et al. Annu Rev Biomed Eng. 2009 Protein identification Single-step? Components for separating identifying and quantifying the polypeptides tools for integrating and analysing all the data Two main tracks. 2DE & MS Limited protein purification & peptide MS/MS +/- isotope tagging Efficient MS identification of gel-separated proteins Peptide-mass mapping by MALDI-TOF Peptide sequencing by ESI-MS/MS Yates JR, et al. Annu Rev Biomed Eng. 2009 Thank you Dimitri Raptis & Alexander Kögel www.draptis.eu/proteomics.ppt Proteomics Alexander Kögel & Dimitri Raptis Post-translational modifications using mass spectrometry www.draptis.eu/proteomics.ppt The Mass Spectrometer (MS) Moving ions are separated in a magnetic field according to their mass-charge-ratio Importance for modern Proteomics Molecular weight determination of peptides and proteins Amino acid sequencing of peptides Detection of post-translational modifications (ptm) Protein phosphorylation Phosphoregulation Common regulation of protein function Phosphorylaion -> Activation Mostly Ser, Thr, Tyr are phosphorylated by protein kinases Mann & Jensen, Nature Biotech, 2003 Identification of a Phosphorylation-site 1. Isolation of known protein 2. Enrichment with Metal Affinity Complexation 3. Digestion with Trypsin 4. Mass Spectrometry with peptides Identification of a Phosphorylation-site Modification is proven to be in a peptide of certain mass Identification of a Phosphorylation-site Further fragmentation of peptide in collision chamber Second analysis with MS and comparison to databank will lead to a shifted aa in the MS screen Most studied PTMs Thank you Alexander Kögel & Dimitri Raptis www.draptis.eu/proteomics.ppt