Clinical Proteomics: A T A echnology to shape the future? echnology to

Transcription

1 Clinical Proteomics: A Technology to shape the future? Cyprus, Nov, 2010

2 Protein Chemistry/Proteomics/Peptide Synthesis and Array Unit Institute of Biomedicine/Anatomy Biomedicum Helsinki, University of Helsinki, Finland (

3 Over 1200 Researchers in only Medical Research (Cancer, Genetics, Developmental Medicine, Neuroscience etc.)

4 The Medical Faculty

5

6 Proteomics What is it all about??

7

8 Proteomics in prostate cancer. Banez LL, Srivastava S, Moul JW. Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Rockville, Maryland, USA bdivision of Urology, Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA. Curr Opin Urol May;15(3): Proteomics of disease. Celis JE, Korc M. Mol Cell Proteomics Apr;4(4):345.

9 We know what Genomics is! But, what is Proteomics?

10

11 2001: Human Genome Project Reveals 3,000,000,000 base pair nucleotides = only 25,000 genes =

12 And that Only 0.1% of each s persons DNA differs from any other person =

13 So what makes the Difference? = Proteins The study of the Proteins expressed by a Genome = Proteome

14 same Genome - different Proteome

15 How can we study Proteomics? The technology... 2D gel electrophoresis 2D liquid chromatography Micro arrays

16 An example

17 A technology to find changes in Proteome Administration of a drug known to bind to an orphan receptor Changes in expression level of 23 proteins A protein Array

18 Two-dimensional gel electrophoresis (2D) 1st dimension, IEF, Proteins are separated according to their isoelectric point (IP) 2nd dimension, SDS- PAGE, Proteins are separated according to their molecular mass Efficient: More than a thousand proteins resolved in E-Coli cell lysates and ~8000 in brain lysates ph 3 SDS- PAGE ph 10

19 1 st Dimension - Isoelectric Focusing ph4 ph5.5 ph neutral gelsurface pi - isoelectric point ready made Gel-strips

20 2 nd Dimension - Isoelectric Focusing 2DE Mw

21 Protein Fingerprint: 2-DE 200 Mr (kda) About 2000 proteins pi 6.9

22 2D-PAGE Direct In-situ Digest To identify the separated proteins Basic (+) Acidic (-) #7 100 fmol running the gel - staining each spot of interest ( ) - excise - in-gel digestion PEPTIDE ANALYSIS Trypsin digest

23

24 MALDI TOF/TOF

25 MALDI-TOF-MS High Sample Throughput for the Post-Genomic Era

26 MALDI TOF Schematic diagram of a Reflector MALDI-TOF mass spectrometer. MALDI-TOF = Matrix Assisted Laser Desorption/Ionization - Time Of Flight

27 MALDI TOF/TOF

28 Quadrupole TOF/TOF

29 Quadrupole TOF/TOF

30 Quadrupole TOF/TOF

31 Typical mass spectrum from a protein band trypsin digest

32 Automated Database Search (MASCOT) Number 1 match: tumor necrosis factor type 1 receptor associated protein TRAP-1 (Mr): RALRRAPALA AVPGGKPILC PRRTTAQLGP RRNPAWSLQA GRLFSTQTAE 51 DKEEPLHSII SSTESVQGST SKHEFQAETK KLLDIVARSL YSEKEVFIRE 101 LISNASDALE KLRHKLVSDG QALPEMEIHL QTNAEKGTIT IQDTGIGMTQ 151 EELVSNLGTI ARSGSKAFLD ALQNQAEASS KIIGQFGVGF YSAFMVADRV 201 EVYSRSAAPG SLGYQWLSDG SGVFEIAEAS GVRTGTKIII HLKSDCKEFS 251 SEARVRDVVT KYSNFVSFPL YLNGRRMNTL QAIWMMDPKD VGEWQHEEFY 301 RYVAQAHDKP RYTLHYKTDA PLNIRSIFYV PDMKPSMFDV SRELGSSVAL 351 YSRKVLIQTK ATDILPKWLR FIRGVVDSED IPLNLSRELL QESALIRKLR 401 DVLQQRLIKF FIDQSKKDAE KYAKFFEDYG LFMREGIVTA TEQEVKEDIA 451 KLLRYESSAL PSGQLTSLSE YASRMRAGTR NIYYLCAPNR HLAEHSPYYE 501 AMKKKDTEVL FCFEQFDELT LLHLREFDKK KLISVETDIV VDHYKEEKFE 551 DRSPAAECLS EKETEELMAW MRNVLGSRVT NVKVTLRLDT HPAMVTVLEM 601 GAARHFLRMQ QLAKTQEERA QLLQPTLEIN PRHALIKKLN HCAQASLAWL 651 SCWWIRYTRT P Total coverage: 33.4%

33 Even a single peptide is enough for Protein identification!

34 We can do a peptide amino acid sequence analysis by mass spectrometry!

35 2D-PAGE Direct In-situ Digest (Unknown protein identification) Basic (+) Acidic (-) #7 100 fmol running the gel - staining each spot of interest ( ) - excise - in-gel digestion PEPTIDE ANALYSIS Trypsin digest but take a single peptide to the MS!

36 MALDI MS TOF/TOF or Quadrupole MS TOF/TOF Fragmentation of a single peptide in the Mass Spectrometer >>>> Database search MS-MS >>>> Identification of the whole protein

37 Automated Database Search (MASCOT) Number 1 match: tumor necrosis factor type 1 receptor associated protein TRAP-1 (Mr): RALRRAPALA AVPGGKPILC PRRTTAQLGP RRNPAWSLQA GRLFSTQTAE DKEEPLHSII SSTESVQGST SKHEFQAETK KLLDIVARSL YSEKEVFIRE 101 LISNASDALE KLRHKLVSDG QALPEMEIHL QTNAEKGTIT IQDTGIGMTQ 151 EELVSNLGTI ARSGSKAFLD ALQNQAEASS KIIGQFGVGF YSAFMVADRV 201 EVYSRSAAPG SLGYQWLSDG SGVFEIAEAS GVRTGTKIII HLKSDCKEFS 251 SEARVRDVVT KYSNFVSFPL YLNGRRMNTL QAIWMMDPKD VGEWQHEEFY 301 RYVAQAHDKP RYTLHYKTDA PLNIRSIFYV PDMKPSMFDV SRELGSSVAL 351 YSRKVLIQTK ATDILPKWLR FIRGVVDSED IPLNLSRELL QESALIRKLR 401 DVLQQRLIKF FIDQSKKDAE KYAKFFEDYG LFMREGIVTA TEQEVKEDIA 451 KLLRYESSAL PSGQLTSLSE YASRMRAGTR NIYYLCAPNR HLAEHSPYYE 501 AMKKKDTEVL FCFEQFDELT LLHLREFDKK KLISVETDIV VDHYKEEKFE 551 DRSPAAECLS EKETEELMAW MRNVLGSRVT NVKVTLRLDT HPAMVTVLEM 601 GAARHFLRMQ QLAKTQEERA QLLQPTLEIN PRHALIKKLN HCAQASLAWL 651 SCWWIRYTRT P Sequence successfully identified

38 What can Proteomics give us? A typical case...

39 Colorectal Crypt - Physiology and Transformation Stem cells are located in the lower crypt poles, colonocytes are migrating to the surface of the crypt Continuelly replacement in the toxic and mechanically stressing environment of the gut Hyperproliferation is believed to precede adenoma formation Loss of APC function leads to deregulated crypt homeostasis and is thought to be the initiating event in adenoma formation

40 Correlation of Histological Progression and Molecular Changes in Colorectal Cancer APC, -catenin mutation Normal mucosa K-ras mutation Aberrant crypt foci Early adenoma 18q loss, p53 mutation Late adenoma Nm23 mut. Invasive carcinoma Metastasis

41 Samples Polyp Normal Liver metastasis Carcinoma

42 Tissue microdissection

43 Why Tissue Microdissection? Normal Tissue Tumor Tissue You would like to isolate only the targeted diseased tissue

44 Laser-based microdissection:

45 Laser Capture Microdissection (Arcturus Autopix)

46 Laser Capture Microdissection Emmert-Buck et al (1996) Laser Capture Microdissection Science, Vol 274, Nro 8, p

47 Capturing of the vessels in the control brain slide Cap Slide before capture Slide after capture Cap with the selected tissue *

48 Why Tissue Microdissection? Normal Tissue Tumor Tissue Cryostat section

49 MALDI MS analysis directly from the tissue captured on the cap membrane

50 Electron micrograph, dermal artery with some granular osmiophilic material (GOM) CADASIL disease

51 Scanning/ Gel-Matching /Imageanalysis Normal Polyp Tumor

52 N P T 1 st Dimension M Sample enrichment/ preparation 2-DE Algorithm 2 nd Dimension PC based matching Mass spectrometrical identification Statistics

53 normal metastasis Principal Component Analysis (t1,t2,t3) polyp tumor

54 Deviating Proteins (n=112) Identification of 72 N=26 N=46 N P T M N P T M benign maligne benign maligne Controls: normal liver tissue HCT116 and Lovo cell lines

55 Intra-individual expression differences of Cytokeratin 20 in patient 14. CR174N CR173P CR172T a b c The left gel segment (a) is zoomed from the normal mucosa, gel b represents the patients polyp and gel c is the corresponding segment of the same patients adenocarcinoma

56 Ettan TM DiGE the quantitative approach to do Proteomics

57 CyDye DIGE Fluor dyes Minimal labelling dyes Label 50 µg of protein 3 colors: Cy 2, Cy3, Cy5 MW matched (~450Da) Charge matched (positive) Label -amino group of lysine Sensitivity ng Linear dynamic range over 4 orders of magnitude

58 Achieving accurate quantitative data Ettan DIGE system Pooled internal standard label with Cy 2 Cy2 Protein extract 1 label with Cy3 Cy3 Protein extract 2 label with Cy5 Cy5 Mix labelled extracts 2-DE separation Typhoon Variable Mode Imager DeCyder Differential Analysis Software

59 Overlay of normal and patient protein samples Increased abundance Equal abundance Reduced abundance Normal control = Cy TM 3 labelled - Blue Patient A sample = Cy5 Labelled - Red

60

61 Also 2D Databases exist!

62 Protein Fingerprint: 2-DE 200 Mr (kda) About 2000 proteins pi 6.9

63 An Analytical Challenge = What about having thousands of proteins in the sample and the need for automation? Yeast database search reveals: 6,188 potential proteins tryptic digestion 344,855 peptides

64 2D-LC/MS/MS (2-dimensional liquid chromatography mass spectrometry) Faster Protein Identification by MS/MS

65 2D-LC/MS/MS Instrumental Set-up SCX/RP/RP SCX µ-column 300 µm i.d. x 5 mm Waste Nano LC gradient Loading 20 µl/min µ-precolumn 300 µm i.d. x 1 mm, C8 Sample & Salt Plugs Nano column 75 µm i.d. x 15 cm, C18 zz Waters 2D-nano UPLC aquity LC

66 Loading 20 µl/min Sample Waste 2D-LC/MS/MS Nano LC gradient Instrumental to their Set-up physico-chemical SCX/RP/RP chemical identity zz Each protein/peptide is separated according SCX µ-column 300 µm i.d. x 5 mm Waste Nano LC gradient Loading 20 µl/min µ-precolumn 300 µm i.d. x 1 mm, C8 Sample & Salt Plugs Waters nano-aquity Nano LC system with 2D Nano column 75 µm i.d. x 15 cm, C18 zz

67 We use the property of proteins/peptides to behave differently in different ph

68

69 The time for one analysis is critical?

70 You can analyze thousands of compounds in a few hours

71 You can analyze thousands of compounds in a few hours

72 The whole 2D LC can be done in a chip

73 The whole 2D LC can be done in a chip

74 2D-CE/MS/MS

75 High throughput CE Capillary windows Capillaries LED detection (MIT, Forest et al, 2006) Autosampler

76 Diagno-Proteomics

77 Printing proteins as microarrays for high-throughput function determination Prot G P50 FRB Detecting protein-protein interactions on glass slides BODIPY CY3 CY5 and rapamycin CY5 only Everything together Science Sep 8;289(5485):

78 Printing proteins as microarrays for high-throughput function determination A single slide holding 10,800 spots. Protein G was printed 10,799 times A single spot of FRB was printed in row 27, column 109. The slide was probed with BODIPY, CY3, CY5 and rapamycin Science Sep 8;289(5485):

79 Automated Micro-Chip Robot loader

80 What else can we do?

81 DE C

82

83 2 DE Performance Native IEF and native PAGE 5 variants of hemoglobin ph Native IEF and SDS-PAGE standard IEF proteins ph 3-10 Denatured IEF and SDS-PAGE HbA 2 HbF HbS GFAP protein variants expression differences in control and Alzheimer diseased patients ph 4-6 HbA 1c HbA control AD

84 SELDI = Chip based surface enchanced laser desorption analysis

85

86 The Chip

87

88

89 Current Developments In SELDI Affinity Technology Mass Spectrometry Reviews, 23, 34-44, (2004) High Throughput

90 Micro-Array technology in Proteomics Protein arrays

91 Ab-micro array

92 Protein Array Micro-Array technology in Proteomics

93 Microfluidics in a Chip for Proteomics Biochip-IMB, Ltd.

94

95 Biochip-IMB, Ltd. Express diagnostics tools CUSTOMIZED

96

97 IMS Imaging mass spectrometry

98

99 Principles Tissue section (mouse brain) Ion intensity Acquisition y 12mm Acquisition x 2,000 15,000 30,000 m/z A laser is rastered over a defined area while acquiring a complete mass spectrum from each position, resulting in molecular images for multiple analytes Cornett, et al., Nature Methods 2007

100 IMS workflow Stoeckli, M, et al., Analytical Biochemistry 2002

101 Tissue slide for IMS

102 Benefits of MALDI-IMS IMS Analysis of entire sample in one reading Previous knowledge of molecular composition is not necessary Allows for investigation of disease formation, progression, and treatment

103 Targeted labeling MS imaging advantages No labeling required Biomolecules are functionally unmodified Label free imaging Image biomolecular modifications PTM s, Metabolites Detailed information on molecular identity By 2D MALDI Large scope of different elements and molecules

104 IMS vs. histochemical stain

105 Imaging MS applications Drug administration and detection

106

107 m/z 381 New Phytologist (2007) 173 : m/z 543 m/z 705

108 Reconstruction of the Carbohydrate moiety Of WHEAT (Triticum aestivum)

109 Special Applications

110 Immuno-MS-IMS Imaging of regions immunoreactive with anti-synaptophysin Ab in healthy human pancreas. (A) Localization of synaptophysin positive cells by TAMSIM. The monoclonal rabbit anti-synaptophysin is conjugated with the tag El 307 (498 m/z). The false color green points in the section show the presence of the tag El 307 and thus synaptophysin positive cells. (B) Classical IHC image with the anti-insulin Ab. The dark pink spots correspond to Langerhans islets and so the synaptophysin-positive cells. The distribution of synaptophysin positive cells in (A) is very similar to that in (B). Thiery, G., et al., Proteomics 2008