Protein Microarrays?

Transcription

1 Protein Microarrays? Protein Chemistry/Proteomics Unit and the Neuroscience Program,Biomedicum Helsinki (

2 What is it all about??

3 Microfluidics Fundamentals of microfabrication; Microfluidics Bioanalytical micro and nano devices; Microchip platform development: design, development, and integration of functional elements (sample propulsion elements, microreactors, separation/infusion channels, MS interfaces, filters, mixers, interconnecting units, microdispensing elements, multiplexed architectures, etc.). Bioanalytical process implementation on the chip: sample cleanup, prefractionation, preconcentration, labeling, digestion, and separation. Developing chemistries based on affinity interactions for capturing, purifying, labeling and immobilizing peptide or protein components. Mass spectrometric (ESI, MALDI, SELDI) analysis of peptide/protein samples.

4 Microfluidics Fundamentals of microfabrication; Microfluidics Bioanalytical micro and nano devices; Microchip platform development: design, development, and integration of functional elements (sample propulsion elements, microreactors, separation/infusion channels, MS interfaces, filters, mixers, interconnecting units, microdispensing elements, multiplexed architectures, etc.). Bioanalytical process implementation on the chip: sample cleanup, prefractionation, preconcentration, labeling, digestion, and separation. Developing chemistries based on affinity interactions for capturing, purifying, labeling and immobilizing peptide or protein components. Mass spectrometric (ESI, MALDI, SELDI) analysis of peptide/protein samples.

5 2001: Human Genome Project Reveals 3,000,000,000 base pair nucleotides only 33,000 to 45,000 genes are actively expressed =

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

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

8 Proteomic Research Two main objectives: - Quantification of all the proteins expressed in a cell - Functional study of thousands of proteins in parallel For quantification purposes, standard method is 2DE electrophoresis or MudPIT separation followed by MS identification For protein function studies, microarray based assays are used to study protein-protein and protein-ligand interactions

9 How do we do it and why? Separation and Identification

10 Protein Microarrays

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12 Protein Chemistry Unit Biomedicum Helsinki 2002

13 Protein Chemistry Unit Biomedicum Helsinki 2002

14 Proteomics Microchips Pump Pump IEF PAGE 2,4 cm x 2.4 cm

15 1-D Gel Electrophoresis electrodes gel reservoir: 25x32x0.3/0.5 mm PMMA, silica sample inlet buffer reservoir gel reservoir water cooling analysis device standard real sample

16 Micro 2-Dimensional PAGE/ Micro 1-Dimensional PAGE 2 cm 2,5 cm Running time minutes Running time 10 minutes

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18 NanoSpray LC/MS (MudPIT( MudPIT) ** * * * peptide 1 * peptide 2 * * * *

19 Instrumental Set-up SCX/RP/RP (MudPIT) 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 Esquire

20 NanoSpray LC/MS TriVersa NanoMate and ESI Chip System Advion Biosiences

21 NanoSpray LC/MS

22 Microarray MS Chip (IonTrap( IonTrap)

23 Microarray MS Chip (IonTrap( IonTrap)

24 Microarray MS Chip (IonTrap( IonTrap)

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27 A set of thousands of Mass Spectrometers in one chip

28 On-Line MALDI Protein Microarray 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

29 Automated Micro-Chip Robot loader

30 MALDI-TOF-MS High sample throughput for the Post-Genomic Era

31 What else can we do?

32 SELDI = Chip based surface enchanced laser desorption analysis

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34 The Chip

35 DIOS-MS Desorption Ionization On Silicon (DIOS) laser MS porous area sample

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38 PRIMER ON THE SELDI PROCESS Step One: Choosing an array ProteinChip Arrays are available with different chromatographic properties, including hydrophobic, hydrophilic, anion exchange, cation exchange, and immobilized-metal affinity surfaces. Other ProteinChip Arrays with pre-activated surfaces are available for covalently coupling protein, DNA, RNA or other bait molecules by the user

39 Step two: Sample application Crude biological samples such as serum, cell lysates or other protein preparations, including those with high salt or detergent concentrations, can be applied directly to the ProteinChip Arrays. Application can be done manually by pipetting or by employing Ciphergen s customized configuration of the Beckman Coulter Biomek 2000 laboratory automation station. The arrays are formatted with robot-friendly spot spacing and a bioprocessor rack of 12 arrays forming a standard microplate footprint.

40 Step three: Removal of unbound components After a short incubation period, unbound proteins are washed off the surface of the ProteinChip Array. Only proteins interacting with the chemistry of the array surface are retained for analysis. After washing, energy absorbing molecules are applied to the array as a final step.

41 Step four: Analysis in the ProteinChip Reader ProteinChip Arrays are then analyzed in the ProteinChip Reader, a time-of-flight mass spectrometer. The mass values and signal intensities for the detected proteins and peptides can be viewed in several formats and then transferred to Ciphergen s powerful software suites for further in-depth analysis.

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

47 Unraveling biological pathways using the Interaction Discovery Mapping platform Whole Cell analysis

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

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50 Biochip-IMB, Ltd. Express diagnostics tools CUSTOMIZED

51 Direct on-chip mass-spectrometry Protein-ligand interactions Biotinylated proteins are bound to avidin immobilized in gel pad.

52 Some Chip structures

53 ESI chip geometry Electrospray orifice Huikko et al., Lab.Chip., 2003

54 A typical Chip and how to Make it... Experion Automated Electrophoresis Station

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56 Protein Microarray Chip structures Development of Integrated Nanoliter Analysis Devices (DDTC-Viikki, Microtechnology Center- HUT, Biomedicum Helsinki)

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58 µapci source vs. macroapci source (PE Sciex API-300)

59 Silicon etching -anisotropic wet etching (100), (110), (111) -plasma etching/drie -electrochemical etching

60 What else do we have now? Affinity based protein fishing Chips: Protein Chips e.g. from several companies

61 Micro-Array technology in Proteomics Protein arrays

62 Ab-micro array

63 Protein Array Micro-Array technology in Proteomics

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65 The MEMS = micro-electromechanical systems 50um gears for a missile

66 A micromirror in a micro computer display (TI) One sugar molecule

67 Airbag in a Car

68 We want an integrated Nanoliter Analysis Device

69 Gel electrophoresis on a capillary in one of the channels (500um) 1 mm

70 Pocket Laboratory

71 Air Bubble Control System

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73 The Factory... The Goal in Microfabrication...?

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