1. Proteomics and Mass Spectrometry Ron Bose, MD PhD Biochemistry and Molecular Cell Biology Programs Washington University School of Medicine Molecular Cell Biology Lecture. Oct. 15, 2015

2. Introduction Well Established Methods for Proteomics 1.2D-gels 2.Mass Spectrometry Methods still under development 1.Protein Arrays 2.Antibody Arrays 3.Proteome-wide coverage with Antibodies Definition of Proteomics: The large scale identification and characterization of proteins in a cell, tissue, or organism.

3. 2 Dimensional Gel Electrophoresis First Dimension: pI by Isoelectric Focusing Second Dimension: MW by standard SDS-PAGE First Published in 1975 by Pat O’Farrell Can separate at least 1,000 proteins Problems with run to run reproducibility limits the ability to easily compare multiple samples. Solution to this problem: DIGE (Difference Imaging Gel Electrophoresis) Size Charge (pI)

4. DIGE experiment Slide courtesy of Tracy Andacht

5. 1.Protein solubility during Isoelectric Focusing. Membrane proteins often lost. 2.Size Limits – difficulty with proteins >100 kD. 3.Identification of the proteins in each spot is tedious and slow. Use of robotics 4.Individual spots typically contain several proteins. Intensity change is therefore the sum of the changes of each individual protein. Limitations of DIGE

6. The Importance of Mass: 1.The mass of a molecule is a fundamental physical property of a molecule. 2.Mass can be used to identify the molecule. Fragmentation provides Chemical Structure: If you fragment a molecule in a predictable manner and make measurements on the individual fragments, you can determine the chemical structure of the molecule. Principles of Mass Spectrometry

7. Trypsin – a protease that cleaves after basic residues (R or K). Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link Protein of Interest:

8. Products from Trypsin digest. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link Average length of tryptic peptides = 10 aa residues

9. Select an Individual Peptide in the Mass Spectrometer Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link Performed by adjusting the electrical fields in the mass spectrometer.

10. Impart energy to the peptide by colliding it with an inert gas (Argon or Helium). Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link

11. Measure the masses of the fragment ions. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link

12. The mass difference between the peaks corresponds directly to the amino acid sequence. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link B -ions contain the N- terminus

13. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link Y -ions contain the C-terminus

14. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link The entire spectrum contains B -ions,Y -ions, and other fragment ions.

15. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link The puzzle: The B, Y, and other ions occur together and we cannot distinguish them just by simple inspection of the spectrum.

16. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link Actual spectra also have noise (either chemical noise or electrical noise).

17. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Slide courtesy of Andrew Link The final spectrum: the interpretation requires experience and aid by software algorithms. Final spectrum: 1.B ions 2.Y ions 3.Other fragments 4.Noise

18. Y8Y8 Y6Y6 Y7Y7 Y4Y4 Y5Y5 Y3Y3 Y2Y2 Y1Y1 L V Q I G D D Peptide 326-334 with phosphorylation on Y326 B3B3 B2B2 pYLVIQGDDR Example of an Actual Spectrum pY Imm.

19. Limitations and Cautions of Proteomics: The Range of Protein Concentrations In Human Plasma Anderson & Anderson, MCP 1:845, 2002 3 - 4 log range of Mass Spectrometers Albumin 40 g/lC4 Complement 0.1 g/l Myoglobin < 100  g/lTNF  < 1 ng/l

20. Limitations and Cautions of Proteomics: The Range of Protein Concentrations In Human Plasma Depletion of unwanted proteins Remove abundant proteins that are not of interest to your experiment. Methods: Antibody based depletion, selective lysis technique, subcellular fractionation, etc. Enrichment Enrich for the proteins of interest. Methods – Lysis techniques or subcellular fractionation, affinity-based enrichment (antibodies, resins, etc). Fractionation of sample to divide sample Reduce the complexity of your sample by separating the proteins into different fractions and running these fractions separately.

21. Amine reactive tags – iTRAQ (Ross et al., MCP 3:1154, 2003) Cys reactive tags - ICAT Incorporating 18 O during Trypsin digestion Protein Quantification with Mass Spectrometry Introduce Stable Isotope by Chemical Labeling

22. Studying EGFR Signal Transduction with Quantitative Proteomics Introduce Stable Isotope by Chemical Labeling Zhang et al., MCP 4: 1240-50, 2005

23. Mapping Her2/neu Tyrosine Kinase Signaling using Quantitative Proteomics 250 kD 150 kD 100 kD 75 kD Vehicle 0.010.118 Her2 inhibitor (  M) Gefitinib 1  M Empty Vector Her2/neu A.B. Bose et al., PNAS 103:9773, 2006 WB: Anti-pTyr

24. Empty vector cells Her2/neu cells +Her2 kinase inhibitor Her2/neu cells Mix Lysates Immunoaffinity Purify with Antiphosphotyrosine Antibodies Resolve on SDS-PAGE Digest Bands with Trypsin Identify and Quantify Proteins by LC-MS/MS Bose et al., PNAS 103:9773, 2006 Mapping Her2/neu Tyrosine Kinase Signaling using Quantitative Proteomics Introduce Stable Isotope by Metabolic Labeling

25. 500.0502.0504.0506.0508.0 505.303 503.309 500.304 Protein 1 635.0637.0639.0641.0643.0 635.924 640.925 638.930 Protein 3 637.0639.0641.0643.0 645.0 642.405 640.412 637.405 Protein 2 Protein 4 +0 +6 +10 +0 +6 +10 VGQAQDILRVAGQSSPSGIQSR FFEILSPVYRHDGAFLIR Key +0 Control 12 C-Arginine +6 Treatment 1 13 C 6 -Arginine +10 Treatment 2 13 C 6 15 N 4 -Arginine Protein Quantitation with Mass Spectrometry Bose et al., PNAS 103: 9773-8, 2006

26. Signal Transduction Pathway Smorgasbord Ron Bose, MD PhD Biochemistry and Molecular Cell Biology Programs Washington University School of Medicine Molecular Cell Biology Lecture. Oct 27, 2015

27. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

28. Nuclear Hormone Receptor Superfamily 1.48 Human genes 2.Major Categories: Knock-out in mice causes reproductive, developmental, or metabolic abnormalities. Thyroid Hormone Receptor (TR)- like TR, RAR, PPAR, Vitamin D receptor, LiverX Receptor Estrogen Receptor (ER)- like ER, PR, AR, Estrogen Receptor Related, Glucocorticoid receptor, Mineralocorticoid receptor Retinoid X Receptor (RXR) like RXR, Hepatocyte nuclear factor-4, etc.

29. AF: Activation Function. Mediate transcriptional activation DNA Binding Domain Ligand Binding Domain www.nursa.org/

30. Bind as homodimers Bind as heterodimers with RXR Hormone response elements are inverted repeats. Hormone response elements are direct repeats.

31. Ligand Present Ligand Absent www.nursa.org/

32. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

33. Cytokine Receptors – JAK/STAT Pathway Baker et al., Oncogene (2007) 26, 6724–6737

34. JAK = Janus kinases Baker et al., Oncogene (2007) 26, 6724–6737 4 genes in humans and mice TYK2 (first gene in this family to be identified) JAK1, JAK2, JAK3

35. Cytokine Receptors – JAK/STAT Pathway Baker et al., Oncogene (2007) 26, 6724–6737

36. from Marmor, Skaria, and Yarden 2004 Recptor Tyrosine Kinases Examples– EGFR, Her2, etc

37. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

38. PI3-kinase – Akt PtdIns(4,5)P 2 (PIP2) PtdIns(3,4,5)P 3 (PIP3) PI3K PTEN Akt PDK1 Zoncu et al., Nature Rev Mol Cell Bio 2011

39. mTOR complexes mTORC1 Rapamycin sensitive Responds to nutrient level, growth factors, energy, and stress. mTORC2 NOT rapamycin sensitive Inputs into mTORC2 less well known. Zoncu et al., Nature Rev Mol Cell Bio 2011

40. mTORC1 substrates S6 kinase 1 (S6K1) eIF-4E binding protein (4E-BP) Zoncu et al., Nature Rev Mol Cell Bio 2011

41. mTORC2 substrates Akt Zoncu et al., Nature Rev Mol Cell Bio 2011 PH domain Kinase Domain PDK1 mTORC2 S473T308 Downstream substrates: TSC complex, PRAS40, etc. 1 3 2

42. Bringing it all together Zoncu et al., Nature Rev Mol Cell Bio 2011 mTOR is a signal integrator, like the chips and circuits in your smart phone

43. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

44. Regulation of Protein Kinases 1.Post-translation modifications.  Phosphorylation-dependent  Activation Loop 2.Protein-protein interactions  Regulatory Subunits  Dimers

45. Phosphorylation of the MAP Kinase activation loop Phosphorylation on threonine and tyrosine Phospho-Thr 183 contacts  -C and promotes active conformation Phospho-Thr 183 promotes ERK2 dimerization via conformational changes in C-terminal extension Illustration taken from Huse and Kuriyan, Cell 109, 275-282 (2002) AKT phosphorylation at T308 is also Activation Loop Phosphorylation

46. Asymmetric Dimer Formed by the EGFR Kinase Domain EGFR kinase domain asymmetric dimer Zhang, Gureasko, Shen, Cole, and Kuriyan. Cell 2006

47. Summary 1.Nuclear hormone receptors consist of a DNA- binding domain and ligand-binding domain. 2.Cytokine receptors signal through the JAK kinases, which have 2 kinase domains, and the STAT transcription factors. 3.mTOR is a signal integrator for metabolic and growth factor signaling. 4.Protein kinases are regulated by PTM’s and protein-protein interactions.

48. Signal Transduction Pathways in Human Diseases Ron Bose, MD PhD Biochemistry and Molecular Cell Biology Programs Washington University School of Medicine Molecular Cell Biology Lecture. Oct 29, 2015

49. Introduction Pathways 1.Receptor Tyrosine Kinase 2.G-Protein signaling 3.Cyclic AMP and other Second Messenger Pathways 4.Nuclear Hormone Receptors 5.Cytokine receptors and JAK- STAT pathway Human Diseases Cancer Heart Disease Blood Pressure Kidney Diseases Pain and Pain relief

50. ErbB1 HER1 EGFR ErbB2 HER2 Neu ErbB3 HER3 ErbB4 HER4 I II III IV I II III IV I II III IV I II III IV Extracellular Cytoplasmic Transmembrane Kinase Domain N C N N C C C-terminal tail Zahnow, C.A., Expert Rev Mol Med. 2006 The EGFR family of Receptor Tyrosine Kinases { EGFRHer2/neuHer3Her4 Kinase Domain Inactive

51. Her2/neu and Breast Cancer Her2 first identified as an oncogene from a carcinogen-induced rat brain tumor model. Her2 is gene amplified in about 25% of human breast cancers. Overexpression of Her2 in the mammary gland of transgenic mice causes breast cancer. Herceptin, a monoclonal antibody to Her2/neu, effectively treats Her2 gene amplified human breast cancer.

52. Drugs to Target Receptor Tyrosine Kinases HER2 EGF R HER2 HomodimerHeterodimer Extracellular domain Tyrosine- kinase domains Monoclonal Antibodies ATP-mimetic Tyrosine Kinase Inhibitors

53. ErbB2 Cho et al. (2003) Nature Herceptin Franklin et al. (2004) Cancer Cell ErbB2Pertuzumab Therapeutic Antibodies Target Her2

54. Alterations of ErbB receptors in Cancer Overexpression/Gene Amplification Her2/neu - 25% of breast cancers, rarer in gastric and ovarian cancers. EGFR – many cancers including lung, breast, GI, ovarian, brain Mutations EGFR del E746-A750 EGFR L858R Both found in Lung cancer cases from Non-smokers. These cancers respond very well to Gefitinib (an anti- EGFR tyrosine kinase inhibitor).

55. Chronic Myeloid Leukemia (CML) and the Philadelphia Chromosome CML is diagnosed in 5,000 new patients each year in the US. A classic chromosomal rearrangement between chromosomes 9 and 22, named the Philadelphia chromosome, defines CML. This 9;22 translocation produces a fusion protein between the ABL tyrosine kinase and the BCR gene.

56. Abl can be inhibited with tyrosine kinase inhibitors. –Imatinib (Gleevec) –Dasatinib –Nilotinib Tyrosine kinase inhibitors have revolutionized the treatment of CML, reducing the death rate to only 270 deaths in the U.S. in 2011 (about 5% of incidence rate). Crystal structure of ABL tyrosine kinase with Imatinib (orange) bound. Chronic Myeloid Leukemia (CML) and BCR-ABL Druker, Blood 2008 and Schindler et al., Science 2000.

57. The RARα Nuclear Hormone Receptor in Acute Promyelocytic Leukemia (APL) APL has a characteristic translocation 15;17 that forms the PML-RARα fusion protein. Retinoic Acid (RA) binding converts PML-RARα from a transcriptional repressor to a transcriptional activator. All-trans retinoic acid (ATRA) has made APL the most treatable and best prognosis form of adult acute leukemia. PML Retinoic Acid Binding DNA Binding Retinoic Acid Receptor α ATRA

58. Signaling in Heart and Kidney Diseases  -Adrenergic signaling via GPCR’s 2.Renin-Angiotensin signaling via the Angiotensin II receptor 3.Erythropoietin signaling in anemia of chronic renal disease.

59. Adrenergic Receptors bind Adrenaline/Epinephrine Glycosylation There are also  2 and  3 receptors.

60.  -Adrenergic Receptors stimulate cAMP production GsGs GsGs Adenylate Cyclase cAMPATP Protein Kinase A (PKA)

61. Physiologic Effects of  -Adrenergic Signaling 1.Heart – increased heart rate and contractility 2.Vascular - Dilation of the coronary arteries and arteries to skeletal muscles. 3.Dilation of the airways in the lung Commonly used medications:  -Blockers: control heart rate and blood pressure. Albuterol – The most common medicine for asthma. It is a  2 adrenergic agonist.

62. Downregulation/Desensitization of  -Adrenergic Receptor McDonald and Lefkowitz,Cell Signaling 2001

63. GPCR signaling Controls Blood Pressure via the Renin-Angiotensin System Angiotensinogen Angiotensin I Angiotensin II Renin (kidney) ACE (lung) (Angiotensin Converting Enzyme) Angiotensin II Receptor (GPCR) T ACE inhibitors T Angiotensin Receptor Blockers Common Blood Pressure Medicines

64. GPCR signaling Controls Blood Pressure via the Renin-Angiotensin System Timmermanns, Pharm Review 1993

65. Erythropoietin (EPO) binds to a Cytokine Receptor Nucleus STAT5 DNA JAK2 Tyr Kinase EPO receptor Munugalavadla and Kapur, Reviews in Onc-Hem, 2005

66. EPO Deficiency causes Anemia of Chronic Kidney Disease Chronic kidney disease causes a fall in EPO secretion and this results in decreased red blood cell production (i.e.- anemia). Therefore patients with chronic kidney disease are given recombinant EPO to prevent anemia. Kidney Bone Marrow Increased Red Blood Cell Production EPO

67. Summary 1.Signaling pathways play central roles in cell functioning and in human physiology. 2.Altered signaling pathways cause or contribute to human diseases. 3.Many drugs that are given to people directly affect signal transduction pathways.