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Introduction to Protein Chemistry

Introduction to Protein Chemistry. Gustavo de Souza IMM, OUS. October 2013. Relevance of the Proteome. Relevance of the Proteome. «The recipe of life». X. Chocolate cake : Egg Flour Sugar Baker’s yeast Chocolate. Biological relevance lies on how genes

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Introduction to Protein Chemistry

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  1. Introduction to Protein Chemistry Gustavo de SouzaIMM, OUS October 2013

  2. Relevance of the Proteome

  3. Relevance of the Proteome «The recipe of life» X • Chocolatecake: • Egg • Flour • Sugar • Baker’syeast • Chocolate Biological relevance lies on how genes are expressed and translated to proteins, not if genes are present or not

  4. Amino acid structure

  5. AA side chains

  6. Protein Translation

  7. Peptide Bond

  8. Primary Structure

  9. Primary Structure >sp|F2Z333|CA233_HUMAN Fibronectin type-III domain-containing transmembrane protein C1orf233 MRAPPLLLLLAACAPPPCAAAAPTPPGWEPTPDAPWCPYKVLPEGPEAGGGRLCFRSPAR GFRCQAPGCVLHAPAGRSLRASVLRNRSVLLQWRLAPAAARRVRAFALNCSWRGAYTRFP CERVLLGASCRDYLLPDVHDSVLYRLCLQPLPLRAGPAAAAPETPEPAECVEFTAEPAGM QDIVVAMTAVGGSICVMLVVICLLVAYITENLMRPALARPGLRRHP

  10. Folding

  11. Primary Structure - Folding >sp|F2Z333|CA233_HUMAN Fibronectin type-III domain-containing transmembrane protein C1orf233 MRAPPLLLLLAACAPPPCAAAAPTPPGWEPTPDAPWCPYKVLPEGPEAGGGRLCFRSPAR GFRCQAPGCVLHAPAGRSLRASVLRNRSVLLQWRLAPAAARRVRAFALNCSWRGAYTRFP CERVLLGASCRDYLLPDVHDSVLYRLCLQPLPLRAGPAAAAPETPEPAECVEFTAEPAGM QDIVVAMTAVGGSICVMLVVICLLVAYITENLMRPALARPGLRRHP

  12. Folding Proteins can adopt only a limited number of different protein folds

  13. Secondary Structure

  14. Tertiary Structure

  15. Quaternary Structure

  16. Primary to Quaternary

  17. Primary to Quaternary

  18. What is a «protein sample» in proteomics? RNA-binding protein modules

  19. Take home message • Proteins are the functionally active molecule in a cell. • They possess a high degree of chemical and structural heterogeneity. • 3. Heterogeneity interfere in how a protein sample can be analyzed

  20. Challenges in Protein and Proteomic Analysis Gustavo de SouzaIMM, OUS October 2013

  21. A dangerous idea… One gene, one protein Homo sapiens

  22. Complexity of Protein Samples in Eukaryotes

  23. Complexity of Protein Samples in Eukaryotes

  24. A less dangerous idea One gene, some proteins (let’s say average 5 per gene) Homo sapiens

  25. Complexity of Protein Samples in Eukaryotes PTMs (modifications thatcontrol conformationchanges in histones)

  26. An even less dangerous idea One protein, possible 8 modification sites Homo sapiens

  27. An even less dangerous idea

  28. But in reality… • One specific cell does NOT express all genes at once! • Several transcriptomics studies indicated that the cells under study have ~14000 transcripts at a certain time Homo sapiens

  29. Proteome Dynamics B C A Genome is a relatively static element of an organism, the proteome is changing accordingly to cell type, cell stage developmet, response to stress, etc.

  30. Proteome dynamics within the same cell Proteome can change with the least of the stimuli within a cell

  31. Proteome chemical heterogeneity DNA - Negatively charged molecule Has the same phisico-chemical featuresregardless of: its nucleotide sequence,its tissue source, its donor source, thespecies of the donor, etc.

  32. Amino acid structure

  33. AA side chains

  34. Proteome chemical heterogeneity Membrane proteins

  35. Proteome dynamic range Genome Mostly, individual genes are observed equimolar amounts in a DNA molecule Transcription/Translation Protein concentration within a cellis unique to each individual protein Difference between most and leastabundant molecule = dynamic range

  36. Proteome dynamic range

  37. Proteome dynamic range Dynamic range of a proteome estimated to be around 10e8 (in serum isbelieved to be over 10e10) Geiger et al., MCP 2012

  38. Proteome dynamic range Difference between the most and lowest abundant proteins Cytoskeleton (Actin, tubulin, vimentin) Protein abundance Chaperons (hsp60, hsp70, calreticulin) Metabolism (glycolisis, ribosomal) Mytochondria (respiratory chain) Structure Nucleus (histones) Organelles Signalling pathway proteins, transcription factors, etc Protein GO classification

  39. Instrumentation Aebersold & Mann, Nature 2003

  40. Instrumentation • Instrumentations with different hardware generate different types of raw data. • Different brands developed different computer formats, with need for different libraries to read the file. • Which lead to development of a whole bunch of specific software usingspecific computational protocols. • Lack of standard routine.

  41. Take home message • Proteomic composition is at least 6x more complex than the genomic composition of a cell, if only number of entities is considered. • It is an ever changing feature, limited by spatial and time constrains. • Chemical properties and dynamic range has an relevant impact in success rate of identification using proteomic methods. • Instrumentation and Analysis is not standardized.

  42. Introduction to Mass SpectrometryInterpreting peptide/protein data Gustavo de SouzaIMM, OUS October 2013

  43. Lets talk about…physics 3D Quadrupole ion trap Linear Quadrupole ion trap

  44. What is it? • Instrument which can detect the mass-to-charge (m/z)of ions (or ionized molecules). • Ionization must generate ions in gas-phase • Ion detection is proportional to its abundance in the sample • MS performs at extremely low pressures (vacuum) • - Any molecule is ionizable: small organic/inorganic chemicals(less than 300 Da), average sized peptides or DNAfragments, intact proteins.

  45. Mass Spectrometry Scheme Ion Source Mass Analyzer Inlet Detector LC MALDI ES Time-of-Flight Quadrupole Ion Trap

  46. Ion Intensity = Ion abundance

  47. Isotopes Normally observed in nature. Mass difference = 1 Da

  48. What to expect from a mass spectrum Intensity m/z Avogadro number = 6.022x10e23 /mol

  49. Peptide mass spectrum • Isotopes (12C, 13C, 14N, 15N)

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