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

2. Relevance of the Proteome

3. «The recipe of life» X Chocolate cake: -Egg -Flour -Sugar -Baker’s yeast -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. > 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

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

19. Take home message 1.Proteins are the functionally active molecule in a cell. 2.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 October 2013 Gustavo de Souza IMM, OUS

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

22. 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 that control conformation changes 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 A BC 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 features regardless of: its nucleotide sequence, its tissue source, its donor source, the species 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 cell is unique to each individual protein Difference between most and least abundant molecule = dynamic range

36. Proteome dynamic range

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

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

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 using specific computational protocols. -Lack of standard routine.

41. Take home message 1.Proteomic composition is at least 6x more complex than the genomic composition of a cell, if only number of entities is considered. 2.It is an ever changing feature, limited by spatial and time constrains. 3.Chemical properties and dynamic range has an relevant impact in success rate of identification using proteomic methods. 4.Instrumentation and Analysis is not standardized.

42. Introduction to Mass Spectrometry Interpreting peptide/protein data October 2013 Gustavo de Souza IMM, OUS

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

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

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

47. Ion Intensity = Ion abundance

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

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

50. - Isotopes ( 12 C, 13 C, 14 N, 15 N) Peptide mass spectrum

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

52. How is a sample ionized? -Electron ionization -Chemical ionization -Fast Atom/Ion Bombardment -Field desorption -Plasma Desorption -Laser Desorption and MALDI -Thermospray -Electrospray -Atmospheric pressure chemical ionization

53. Matrix Assisted Laser Desorption Ionization

54. Peptide spectrum on MALDI

55. Protein spectrum on MALDI

56. A little history… 1985 – First use: up to a 3 kDa peptide could be ionized 1987 – Method to ionize intact proteins (up to 34 kDa) described Instruments have no sequence capability 1989 – ESI is used for biomolecules (peptides) Sequence capability, but low sensitivity 1994 – Term «Proteome» is coined 1995 – LC-MS/MS is implemented «Gold standard» of proteomic analysis

57. A little history… -Laborious -Low reproducibility -Time consuming -Low sensitivity -Limited amount of identifications

58. Gradient elution:200 nl/min Column (75 mm)/spray tip (8 mm) Reverse-phase C18 beads, 3 mm Platin-wire 2.0 kV Sample Loading:500 nl/min No precolumn or split ESI 15 cm Fenn et al., Science 246:64-71, 1989. Electrospray Ionization

59. ESI multiple charged elements Peptides + + (-NH2) + + + Proteins + + +++ ++ ++ + +++ ++ + + ++ + + + ++

60. ESI multiple charged elements + + ++ + + + ++ m/z Intensity 500.5 (+2) 334.0 (+3) 250.75 (+4) 1000 Da

61. 0.5 Da (+2)0.33 Da (+3) Mr = 2297.14 Da Peptides on ESI

62. ESI of intact protein *

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

64. How is an ion mass measured? Time-of-flight m/z

65. How is a ion mass measured? Quadrupoles (RF)

66. How is a ion mass measured? Orbitraps

67. Tandem Mass Spectrometry Inlet Ion Source Mass Analyzer Detector Ion Source Mass Analyzer Detector Mass Analyzer Mass Analyzer Collision cell

68. Data Dependent Acquisition 899.013 MS1 (or MS) MS2 (or MS/MS) *

69. Important Parameters in MS - Resolution - Sensitivity - Dynamic Range… m/z

70. 2+ High resolution in MS 1. mass accuracy Expected mass Observed mass

71. High resolution in MS 1. mass accuracy Av. = 65.8 ppm ± 71.5 Av. = 16.5 ppm ± 11.2 Av. = 2.1 ppm ± 1.9 Av. = 0.68 ppm ± 0.47

72. RT m/z RT m/z 2+ 3+ 2. Peak separation High resolution in MS

73. LC-MS/MS

74. With all we (hopefully) learned so far 1) Use strong detergent for cell lysis and protein solubization (SDS, Triton, NP40, Tween) 2) LysC (cuts C-terminal side of K) and/or Trypsin (C-terminal of K and R)

75. With all we (hopefully) learned so far ADFFFSTTHAASRMSHHHGTYYPPHKRFSDDDDT ADFFFSTTHAASR MSHHHGTYYPPHK FSDDDT ++ ArgLys

76. With all we (hopefully) learned so far 3) Nano-LC (300nL/min) 5) Quadrupole-Orbitrap (QExactive)

77. With all we (hopefully) learned so far Mobile phase A A = 5% organic solvent in water B = 95% organic solvent in water B C18 column, 25cm long Time 20 s

78. 899.013 With all we (hopefully) learned so far MS1 (or MS) MS2 (or MS/MS)

79. With all we (hopefully) learned so far Quadrupole Orbitrap

80. From Michalski et al., MCP 10, 2011. With all we (hopefully) learned so far 172,800

81. Take home message - Great diversity of hardware and principles. Different forms of Ionization and Mass measurement. - For protein ID, information regarding the mass of a integral peptide and the mass of its fragments is enough to provide identification - Mass spectrometry is used to analyze the molecular mass of molecules.